Allosteric modulators of the NMDA receptor and their use in the treatment of CNS disorders and enhancement of CNS function

ABSTRACT

Novel compounds and compositions for modulating NMDA receptor function comprising Conantokin-G and derivatives thereof; methods for modulating NMDA receptor function and methods for treating neuropsychopharmacological disorders, using the novel compounds and compositions of the invention; and a method for screening compounds capable of binding to a novel allosteric modulatory site, are described.

This application is a divisional of U.S. application Ser. No.08/413,490, filed Mar. 30, 1995 now U.S. Pat. No. 5,854,217, which is acontinuation-in-part of U.S. application Ser. No. 08/323,436, filed Oct.14, 1994, now U.S. Pat. No. 383,0998 which is a continuation-in-part ofU.S. application Ser. No. 07/952,818, filed Sept. 28, 1992, abandonedthe disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to compounds and compositions which modulate theNMDA receptor and, more specifically, which modulate the receptorthrough a novel complex site.

BACKGROUND OF THE INVENTION

The N-methyl-D-aspartate (NMDA) receptor is a postsynaptic, ionotropicreceptor which is responsive to, inter alia, the excitatory amino acidsglutamate and glycine and the synthetic compound NMDA, hence thereceptor name. The NMDA receptor controls the flow of both divalent(Ca⁺⁺) and monovalent (Na⁺, K⁺) ions into the postsynaptic neural cellthrough a receptor associated channel. Foster et al. , "Taking apartNMDA receptors", Nature, 329:395-396, 1987; Mayer et al., "Excitatoryamino acid receptors, second messengers and regulation of intracellularCa²⁺ in mammalian neurons," Trends in Pharmacol. Sci., 11:254-260, 1990.

The NMDA receptor has been implicated during development in specifyingneuronal architecture and synaptic connectivity, and may be involved inexperience dependent synaptic modifications. In addition, NMDA receptorsare also thought to be involved in long term potentiation, CentralNervous System (CNS) plasticity, cognitive processes, memoryacquisition, retention, and learning. Furthermore, the NMDA receptor hasalso drawn particular interest since it appears to be involved in abroad spectrum of CNS disorders.

For instance, during brain ischemia caused by stroke or traumaticinjury, excessive amounts of the excitatory amino acid glutamate arereleased from damaged or oxygen deprived neurons. This excess glutamatebinds to the NMDA receptor which opens the ligand-gated ion channelthereby allowing Ca⁺⁺ influx producing a high level of intracellularCa⁺⁺ which activates biochemical cascades resulting in protein, DNA, andmembrane degradation leading to cell death. This phenomenon, known asexcitotoxicity, is also thought to be responsible for the neurologicaldamage associated with other disorders ranging from hypoglycemia andcardiac arrest to epilepsy. In addition, there are preliminary reportsindicating similar involvement in the chronic neurodegeneration ofHuntington's, Parkinson's, and Alzheimer's diseases. Activation of theNMDA receptor has been shown to be responsible for post-strokeconvulsions, and, in certain models of epilepsy, activation of the NMDAreceptor has been shown to be necessary for the generation of seizures.

Neuropsychiatric involvement of the NMDA receptor has also beenrecognized. Blockage of the NMDA receptor Ca⁺⁺ channel by the animalanesthetic PCP (phencyclidine) produces a psychotic state in humanssimilar to schizophrenia (reviewed in Johnson et al., "Neuropharmacologyof Phencyclidine: Basic Mechanisms and Therapeutic Potential," Annu.Rev. Pharmacol. Toxicol., 30:707-750, 1990.) Further, NMDA receptorshave also been implicated in certain types of spatial learning. Bliss etal., Nature, 361:31 (1993). Interestingly, both the spatial and temporaldistribution of NMDA receptors in mammalian nervous systems have beenfound to vary. Thus, cells may produce NMDA receptors at different timesin their life cycles and not all neural cells may utilize the NMDAreceptor.

Due to its broad-spectrum of neurological involvement, yet non-universaldistribution, investigators have been interested in the identificationand development of drugs acting at the NMDA receptor. Drugs acting onthe NMDA receptor are, therefore, expected to have enormous therapeuticpotential. For instance, U.S. Pat. No. 4,904,681, issued to Cordi et al.(Cordi I), describes the use of D-Cycloserine, which was known tomodulate the NMDA receptor, to improve/enhance memory and to treatcognitive deficits linked to a neurological disorder. D-Cycloserine isdescribed as a glycine agonist which binds to the strychnine-insensitiveglycine receptor.

U.S. Pat. No. 5,061,721, issued to Cordi et al. (Cordi II), describesthe use of a combination of D-cycloserine and D-alanine to treatAlzheimer's disease, age-associated memory impairment, learningdeficits, and psychotic disorders, as well as to improve memory orlearning in healthy individuals. D-alanine is administered incombination with D-Cycloserine to reduce the side effects observed inclinical trials of D-Cycloserine, mainly those due to itsgrowth-inhibiting effect on bacteria resulting in depletion of naturalintestinal flora. D-Alanine reverses the growth-inhibiting effect ofD-Cycloserine on bacteria. It is also reported that D-Cycloserineactually has partial agonist character.

U.S. Pat. No. 5,086,072, issued to Trullas et al., describes the use of1-aminocyclopropanecarboxylic acid (ACPC), which was known to modulatethe NMDA receptor as a partial agonist of the strychnine-insensitiveglycine binding site, to treat mood disorders including majordepression, bipolar disorder, dysthymia and seasonal effective disorder.It is also therein described that ACPC mimics the actions of clinicallyeffective antidepressants in animal models. Again, in the examplesprovided, the compound was administered ip. In addition, a copendingU.S. patent application is cited that describes that ACPC and itsderivatives may be used to treat neuropharmacological disordersresulting from excessive activation of the NMDA receptor.

None of the foregoing offers, however, a satisfactory mechanism formodulating NMDA receptor function. Since glycine is necessary forreceptor function, compounds modulating the glycine site offer a limitedrange of control. Further, glycine displays only limited sub-typespecificity and compounds modulating the glycine site are expected tobehave similarly.

Development of drugs targeting the NMDA receptor, although desirous, hasbeen hindered because the structure of the NMDA receptor has not yetbeen completely elucidated. It is believed to consist of several proteinchains (subunits) embedded in the postsynaptic membrane. The first twosubunits determined so far form a large extracellular region whichprobably contains most of the allosteric binding sites, severaltransmembrane regions looped and folded to form a pore or channel whichis permeable to Ca⁺⁺, and a carboxyl terminal region with an as yetunknown function. The opening and closing of the channel is regulated bythe binding of various ligands to domains of the protein residing on theextracellular surface and separate from the channel. As such, theseligands are all known as allosteric ligands. The binding of twocoagonist ligands--glycine and glutamate--is thought to effect aconformational change in the overall structure of the protein which isultimately reflected in the channel opening, partially opening,partially closing, or closing. The binding of other allosteric ligandsmodulates the conformational change caused or effected by glutamate andglycine.

A representation of the NMDA receptor showing schematically theprincipal recognition/binding sites which had been elucidated in theliterature is depicted in FIG. 1. The sites marked "Glu" and "Gly" arethe receptor sites for the principal excitatory amino acidneurotransmitters, glutamate and glycine. The glutamate site alsoselectively binds NMDA. Since the binding of glutamate and glycine hasbeen shown to stimulate the flow of Ca⁺⁺ through the channel, glutamateand glycine are said to have a co-agonist (stimulatory) activity.Several competitive inhibitors of the actions of glutamate or glycinealso bind to these sites and include those identified in the boxes inFIG. 1 labeled "NMDA Antagonists" and "Glycine Antagonists." Since thesecompetitive inhibitors of the glutamate site block the flow of Ca⁺⁺through the channel, they are said to have an antagonist activity. Theligand-gated ion channel of the NMDA receptor is, thus, under thecontrol of at least two distinct allosteric sites.

Two subunits of the mouse NMDA receptor channel have been identified bycloning and expression of complementary DNAs designated NR1 and NR2.Four subtypes of NR2 have been identified: NR2a, NR2b, NR2c, and NR2d.The heteromeric NR1/NR2a, NR1/NR2b and NR1/NR2c NMDA receptor channelsexhibit distinct functional properties in affinities for agonists andsensitivities to competitive antagonists and Mg²⁺ block. In contrast tothe wide distribution of the NR1 and NR2a subunit messenger RNAs in thebrain, the NR2b subunit mRNA is expressed only in the forebrain and theNR2c subunit mRNA is found predominantly in the cerebellum. Thesefindings suggest that the molecular diversity of the NR2 subunit familyunderlies the functional heterogeneity of the NMDA receptor channel.Kutsuwada et al, Nature, 358:36-40 (1992).

Several compounds are known which are antagonistic to the flow ofcations through the NMDA receptor but which do not competitively inhibitthe binding of allosteric ligands to any of the known sites. Instead,these compounds bind inside the open cation channel and are generallyknown as channel blockers. These are shown in FIG. 1 in the box labeled"Channel Blockers." In fact, binding of a radiolabeled form of one suchchannel blocker, dizocilpine (i.e., [³ H]MK-801), is a good measure ofthe activation of the NMDA receptor complex. When the channel is open,[³ H]MK-801 may freely pass into the channel and bind to its recognitionsite in the channel. Conversely, when the channel is closed, [³ H]MK-801may not freely pass into the channel and bind. When the channel ispartially open (partially closed) less [³ H]MK-801 is able to bind thanwhen the channel is fully open.

Channel blockers such as MK-801 and antagonists are known to protectcells from excitotoxic death, but in their case the cure may be asundesirable as the death since they block any flux of Ca⁺⁺ therebyeliminating any chance of resumed normal activity. Channel blockers andglutamate site antagonists are known to cause hallucinations, high bloodpressure, loss of coordination, vacuolation in the brain, learningdisability and memory loss. PCP, discussed previously, produces aschizophrenic state in man.

Mg⁺⁺ and Zn⁺⁺ also modulate the NMDA receptor. The exact location of thedivalent cation binding sites is still unclear. Zn⁺⁺ appears to beantagonistic to channel opening and appears to bind to an extracellulardomain. Mg⁺⁺ shows a biphasic activation curve--at low concentrations,it is an agonist for NMDA receptor function and at high concentrationsit is an antagonist. It appears to be absolutely necessary for properreceptor functioning and appears to bind at two sites--a voltagedependant binding site for Mg⁺⁺ within the channel and anothernon-voltage dependent binding site on the extracellular domain. Thesesites are also indicated in FIG. 1 by "Mg⁺⁺ " and "Zn⁺⁺ ".

It is believed that the channel is in constant motion, alternatingbetween a cation passing (open) and a cation blocking (closed) state. Itis not known at present whether the allosteric modulators actuallyincrease the time during which the channel is open to the flow of ions,or whether the modulators increase the frequency of opening. Botheffects might be occurring at the same time. Thus, the terms open andclose, or agonistic and antagonistic, as used herein refer to a timeaveraged affect.

Recently, a third class of agonists which modulate the excitatorysynaptic transmission at the NMDA receptor has been identified. (Ransomet al., "Cooperative modulation of [³ H]MK-801 binding to theN-methyl-D-aspartate receptor-ion channel complex by L-glutamate,glycine, and polyamines," J. Neurochem., 51:830-836, 1988; Reynolds etal., "Ifenprodil is a novel type of N-methyl-D-aspartate receptorantagonist: interaction with polyamines" Molec. Pharmacol., 36:758-765,1989; reviewed in Williams et al., "Modulation of the NMDA Receptor byPolyamines," Life Sci., 48:469-498, 1991.) These agonists arepolyamines, principally the endogenous polyamines spermine andspermidine, which bind to other extracellular allosteric sites on theNMDA receptor. In FIG. 1, the allosteric polyamine binding site islabeled "PA." The polyamines do not bind to the glutamate/NMDA site, theglycine site, or the channel blocker sites. However, polyamines do alsobind inside the channel. There may be some relation between the Mg⁺⁺binding sites and the polyamine sites, but this relationship has not yetbeen fully elucidated. In contrast, there is strong evidenceaccumulating that polyamine binding is not thought to be necessary forfunctioning/activation of the NMDA receptor coupled cation channel, butis necessary for maximum activation. The polyamines, thus, areallosteric modulators of the NMDA receptor.

There appears to be a broad range of polyamine (diamine, triamine, andtetraamine) compounds which will modulate the site, some of which appearto be agonists, others partial agonists, and, finally, some antagonists.Binding of the compound 1,10-diaminodecane (DA10) decreases rather thanincreases the channel opening. This activity has been termed "inverseagonist" activity. Such inverse agonist binds competitively at the samesite as an agonist but produces the opposite effect as the agonist.

Ideally, a drug for regulating the NMDA receptor will modulate theresponse of one of the endogenous ligands, and not itself be anendogenous ligand. The drug must be specific, i.e., it must affect anidentifiable molecular mechanism unique to target cells that bearreceptors for that drug.

It is therefore an object of the present invention to provide a class ofspecific drugs through the discovery of a novel binding site on the NMDAreceptor for a compound which is not an endogenous ligand of thereceptor.

It is a further object of the present invention to provide novelcompounds for regulating the flow of Ca⁺⁺ through the NMDA receptor, andcompositions and methods for treating neurodegenerative disorders linkedto NMDA receptor function.

SUMMARY OF THE INVENTION

Compounds, derived from the snail peptide Conantokin-G, act asallosteric modulators of the NMDA receptor cation channel and haveeffects ranging from inhibitory to partial modulatory to fullystimulatory on the polyamine or a closely associated modulatory site ofthe NMDA receptor. These compounds, therapeutic compositions, and theiruse for 1) treating neurological, neuropsychological, neuropsychiatric,neurodegenerative, neuropsychopharmacological and functional disordersassociated with excessive or insufficient activation of the glutamatesubtype of the NMDA receptor; 2) treating cognitive disorders associatedwith suboptimal activation or deactivation of the glutamate subtype ofthe NMDA receptor; and 3) improving and enhancing memory, learning, andassociated mental processes, are disclosed. Examples of these disordersinclude acute or chronic neurodegenerative diseases, seizures,depression, anxiety, and substance addiction. The compositions can alsobe used to enhance learning and memory.

In one aspect, the compounds have the following formula (Formula I):##STR1## wherein A⁰ is an amino acid selected from the group consistingof natural, modified, or non-natural amino acids;

A¹ is an uncharged, hydrophobic amino acid;

A², A³ and A⁴ are amino acids independently selected from glutamate,aspartic acid, γ-carboxyglutamate (Gla), 3-carboxyaspartic acid,D-glutamate, phosphoserine, or phosphothreonine;

A⁵ is an uncharged, hydrophobic amino acid;

A⁶ is a peptide chain of from about 2 to about 15 amino acids, saidamino acids selected from natural, modified, or non-natural amino acids;

A⁷ is an amino acid selected from the group consisting of natural,modified, or non-natural amino acids;

A⁸ is a basic amino acid selected from lysine or arginine;

A⁹ and A¹⁰ are amino acids selected from the group consisting ofnatural, modified, or non-natural amino acids;

R₁ is H, C1-C6--CO--, -benzoyl, or -benzoyloxy;

R² is H or C1-C6-alkyl;

x^(a) x^(b), X^(c), and x^(d) are independently 0 or 1;

m and n are independently 0 or 1;

provided that m and n may not both be 0; and

pharmaceutically acceptable salts thereof.

It is provided that the compound of Formula I cannot be x^(a), is 0, A¹is glycine, A² is glutamate, A³ is γ-carboxyglutamate, A⁴ isγ-carboxyglutamate, A⁵ is leucine, A⁶ is a peptide chain of 8 aminoacids of the following composition Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg, A⁷is γ-carboxyglutamate, A⁸ is lysine, A⁹ is serine, A¹⁰ is asparagine,and R¹ and R² are H.

It is further provided that the compound of Formula I is preferably notx^(a) is 0, A¹ is glycine, A² is glutamate, A³ is glutamate, A⁴ isglutamate, A⁵ is leucine, A⁶ is a peptide chain of 8 amino acids of thefollowing composition Gln-Glu-Asn-Gln-Glu-Leu-Ile-Arg, A⁷ is glutamate,A⁸ is lysine, A⁹ is serine, A¹⁰ is asparagine, and R¹ and R² are H.

Preferred compounds are compounds of Formula I where A⁶ is a peptidechain consisting of from about 7 to about 9 natural and/or non-naturalamino acids;

R¹ is H;

R² is H; and

X^(a) is 0;

and pharmaceutically acceptable salts thereof.

Further preferred compounds of formula I wherein A₁ and A⁵ are aminoacids selected from the group glycine, alanine, valine, leucine, orisoleucine.

More preferred compounds ate compounds of Formula I wherein:

A⁶ is a peptide chain consisting of about 8 natural amino acids.

Compounds preferred for their antagonistic properties are compounds ofFormula I wherein n is zero.

Compounds preferred for their agonistic properties are compounds ofFormula I wherein m is zero.

Specifically preferred for their modulatory activity are the followingcompounds:

Gly-Glu-Glu-Glu-Leu-Gln-Glu-Asn-Gln-Glu-Leu-Ile-Arg-Glu-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:5);

Tyr-Gly-Glu-Glu-Glu-Leu-Gln-Glu-Asn-Gln-Glu-Leu-Ile-Arg-Glu-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:6);

Ile-Arg-Glu-Lys-Ser-Asn-NH₂ (SEQ ID NO:7);

Glu-Glu-Glu-Leu-Gln-Glu-Asn-Gln-Glu-Leu-Ile-Arg-Glu-Lys-Ser-Asn-NH₂ (SEQID NO:10);

Gly-D-Glu-D-Glu-D-Glu-Leu-Gln-D-Glu-Asn-Gln-D-Glu-Leu-Ile-Arg-D-Glu-Lys-Ser-Asn-NH₂(SEQ ID NO:11);

Gly-Glu-Ala-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:22);

Gly-Glu-Ser-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO: 23);

Gly-Glu-Ser(p)-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:24);

AcTyr-Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH₂(SEQ ID NO:13);

Asn-Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gin-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO: 14);

Asn(GlcNAc)-Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH₂(SEQ ID NO: 15);

Phe-Gly-Glu-Gla-Gla-Leu-GIn-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO: 16);

tBuTyr-Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH₂(SEQ ID NO:20);

Ser-Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:21);

Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Ala-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:35);

Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Ser-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:36);

Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Ser(p)-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:37);

Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Ala-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:38);

Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Uu-Ile-Arg-Ser-Lys-Ser-Asn-NH₂(SEQ ID NO: 39);

Gly-Glu-Gla-Gla-Leu-Gln-Glu-Asn-Gln-Glu-Leu-Ile-Arg-Glu-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:41);

Gly-Glu-Gla-Gla-Leu-Gln-Ala-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:32);

GIy-Glu-Gla-Gla-Leu-Gln-Ala-Asn-Gln-Ala-Leu-Ile-Arg-Ala-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:42);

Gly-Glu-Gla-Gla-Leu-Gln-Ser-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO: 33);

Gly-Glu-Gla-Gla-Leu-Gln-Ser(p)-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:34);

Gly-Glu-Gla-Gla-Leu-Gln-iodoTyr-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:53);

Gly-Glu-Gla-Gla-Leu-Gln-di-iodoTyr-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH₂(SEQ ID NO:54);

Gly-Glu-Gla-Gla-Leu-Gln-Tyr-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO:31);

Gly-Glu-Gla-Gla-Leu-NH₂ (SEQ ID NO:52);

Gla-Lys-Ser-Asn-NH₂ (SEQ ID NO:49);

Ile-Arg-Gla-Asn-NH₂ (SEQ ID NO:50); and

Lys-Ser-Asn-NH₂ (SEQ ID NO:51).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the known binding sites on theNMDA receptor described in the literature indicating both the sites atwhich stimulatory agonists and inhibitory antagonists bind.

FIG. 2 depicts the Con-G allosteric modulatory complex site and theproposed binding of Con-G thereto.

FIGS. 3a and b plot the stimulation of the NMDA receptor in well-washedforebrain membranes by polyamines as indicated by increased [³ H]MK-801binding to the receptor in the nominal absence of endogenous Glu andGly.

FIGS. 4a and b depict the antagonistic effect of Con-G on spermine (4a)and spermidine (4b) induced [³ H]MK-801 binding at varyingconcentrations of Con-G.

FIG. 5 depicts concentration dependent inhibition of the maximumstimulation of [³ H]MK-801 binding to brain membranes produced byspermine and spermidine as the concentration of Con-G increases.

FIG. 6 depicts the inhibition of spermine-enhanced [³ H]MK-801 bindingby Ala7-Con-G and Con-G.

FIG. 7 depicts the non-competitive, concentration dependent, inhibitionof spermine enhanced binding of [³ H]MK-801 by Ala7-Con-G.

FIG. 8 depicts the partial agonist properties of Glu-Con-G and tBu-Tyr⁰-Con-G.

FIG. 9 depicts the partial agonist properties of Ser3-Con-G andSer(p)3-Con-G.

FIG. 10 depicts the agonistic effects of Con-G(14-17), Tyr⁰ -Glu-Con-G,and Glu 14(12-17)-Con-G.

FIG. 11 depicts the noncompetitive inhibition of Glu Con-G enhanced [³H]MK-801 binding to well-washed brain membranes by increasingconcentrations of Con-G.

FIG. 12 depicts the interaction between Glu-Con-G(12-17) and arcaine.

FIG. 13 shows the effect of increasing concentrations of Con-G uponbasal [³ H]MK-801 binding and Gly (10 μM) and Glu (10 μM) stimulated [³H]MK-801 binding to brain membranes.

FIG. 14 depicts the effect of 10 μM Con-G on Gly stimulated [³ H]MK-801binding to brain membranes as a function of increasing Glyconcentration.

FIGS. 15 a and b depict the effects of Ala7-Con-G on basal, Gly-, andGlu-enhanced [³ H]MK-801 binding.

FIG. 16 depicts the effects of Ala7-Con-G on Gly-enhanced [³ H]MK-801binding.

FIG. 17 depicts the effects of Con-G and Ala7-Con-G on NMDA-stimulatedcyclic GMP level in primary cultures of cerebellar granule cells.

FIG. 18 depicts the neuroprotective effect, as percentage protection, ofMK-801 and Con-G on glutamate induced cell death (neurotoxicity) incerebellar granule cell cultures.

FIG. 19 depicts the specific inhibitory effect of ConG (3 μM) onpolyamine stimulation of the NR1/NR2a, NR1/NR2b, and NR1/NR2c subtypesof the NMDA receptor, measured as, the change in potential.

FIG. 20 depicts the relative antagonism of ConG and analogs, representedas concentration of [³ H]MK-801 bound (fmol) for Con G (circle), Ser¹⁴Con G (inverted triangle), Ala⁷ Con G (diamond), and Ala⁷,10,14 Con G(square).

DETAILED DESCRIPTION OF THE INVENTION

During the past few years, a number of unusual polypeptides have beenisolated from the paralytic venoms of the fish hunting cone snails ofthe genus Conus found in the Philippine archipelago. Many of these,designated "conotoxins," have been discovered to affect ion channelfunction. The paralytic α, μ, and ω conotoxins block nicotinicacetylcholine receptors, sodium channels, and voltage sensitive calciumchannels, respectively (reviewed in Olivera et al., "Diversity of Conusneuropeptides," Science, 249:257-263, 1990.).

Non-paralytic peptides from two of the snails, Conus tulipa and Conusgeographus, have particularly unique compositions since they contain theunusual amino acid γ-carboxyglutamate (Gla), Conantokin-T (21 aminoacids from tulipas) containing 4 and Conantokin-G (hereinafter "Con-G")(17 amino acids from geographus) containing 5, and also contain an amidegroup on the carboxyl terminal peptide. The sequence of these aminoacids is shown below with the γ-carboxyglutamate shown in bold:

Conantokin-G:Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH.sub.2(SEQ ID NO: 1)

Conantokin-T:Gly-Glu-Gla-Gla-Tyr-Gln-Lys-Met-Leu-Gla-Asn-Leu-Arg-Gla-Ala-Glu-Val-Lys-Lys-Asn-Ala-NH₂(SEQ ID NO:12)

Conantokin-T (Con-T) and Conantokin-G (Con-G) were recently reported toact as NMDA receptor antagonists. Haack et al., "Conantokin-T (Aγ-carboxyglutamate containing peptide with N-methyl-D-aspartateantagonist activity)," J. Biol. Chem., 265:6025-6029, 1990; Mena et al.,"ConantokinG: a novel peptide antagonist to the N-methyl-D-aspartic acid(NMDA) receptor," Neurosci. Let., 118:241-244, 1990. These two snailpeptides were shown to decrease NMDA induced increases of intracellularCa⁺⁺ levels and to block NMDA induced increases in cGMP levels in bothprimary neuronal cell cultures and brain slice preparations. Mena et al.(1990), supra, noted that the mechanism of antagonism of Conantokin-Gappeared to be different from previously described competitive andnon-competitive NMDA receptor antagonists but did not report or suggestthe mechanism(s) underlying the antagonism of Conantokin-G orConantokin-T to NMDA receptor response.

While Mena et al., supra, had reported that Con-G was an NMDA receptorantagonist which was not competitive with previously known agonists orantagonists, no one had: 1) elucidated the underlying mechanism by whichthe antagonism is mediated; 2) taught how such antagonism can beutilized to control properties of the receptor; or 3) describedaccurately the chemical/biological structural requirements formodulatory activity. It has been discovered that Con-G inhibits thestimulatory effect of polyamines on the NMDA receptor. However, Con-Gdoes not act at the polyamine site alone, but also acts at a new andunique modulatory site. In fact, Con-G bridges both sites whenfunctioning. Thus, the Con-G site is actually a complex including atleast a portion of the polyamine site and a novel site designated hereinas the Con site. (See FIG. 2.)

It has been discovered, by the present inventor, that Con-G acts as anantagonist of the NMDA receptor through its action as a potent,selective, and noncompetitive inhibitor of the polyamine modulatedresponses with a neurochemical profile which is distinct from previouslydescribed polyamine antagonists. It has also been determined that Con-Gbinds at a unique and heretofore unidentified site on the extracellulardomains of the NMDA receptor.

Further, a new class of allosteric modulators has been discovered whichare derived from Con-G and act as partial agonists, full agonists, orfull antagonists of the NMDA receptor at the polyamine site or a siteassociated with the polyamine site, both of which are encompassed withinthe Con-G site. One of these compounds, a four peptideunit--Gla-Lys-Ser-Asn (SEQ ID NO:49)--is a much more potent agonist forthe polyamine site than any known polyamine. Another derivative,substituting a Gla with Ala at amino acid residue 7 in the native Con-Gsequence, is actually a more potent antagonist than Con-G.

As discussed earlier, in Cordi I and II, supra, and Trullas, supra,agents which act as partial agonists of allosteric sites on the NMDAreceptor were used to selectively modulate the receptor's function,apparently without producing the side effects associated with compoundsthat close the NMDA receptor channel. In addition, Cordi I and II andTrullas described that modulation of NMDA receptor activity by partialagonists is useful in the treatment of a wide range of CNS disorders aswell as in enhancement of CNS function.

Specifically, Cordi I and II and Trullas relate to the use of partialagonists of the glycine site to enhance memory, treat learning andcognitive deficits, treat Alzheimers and age associated memoryimpairrnent, treat psychotic disorders, improve memory, and treat mooddisorders. Recent evidence also indicates that certain types of druginduced convulsions are associated with chemical toxicity affecting theNMDA receptor. Greater control over the modulation of the NMDA receptor,as is possible with the compounds described herein, presents one of thebest hopes for dealing with one of the less tractable current societalproblems.

Definitions

The following are definitions of terms as used throughout the presentspecification. These definitions are provided to assist interpretationof, not limit, the invention.

The term "amino acid" as used herein means an α amino acid. Includedwithin this term are natural amino acids, including unusual amino acidssuch as γ-carboxyglutamate, as well as modified and non-natural aminoacids, such as those disclosed in, for example, Roberts et al., ThePeptides, 5:342-429, 1983, the teachings of which are herebyincorporated by reference.

The term "basic amino acid" as used herein means a amino acid as definedabove, in which the side chain has a net positive charge at pH 7.0including, but not limited to, lysine, arginine, and histidine.

The term "uncharged, hydrophobic amino acid" as used herein means an αamino acid with a hydrocarbon side chain, branched or unbranched, of oneto eight carbons.

The abbreviation "Gla" as used herein refers to the amino acidγcarboxyglutamate or γ-carboxyglutamic acid.

The abbreviation "Glu" as used herein refers to the amino acidL-glutamate or L-glutamic acid.

The abbreviation "D-Glu" as used herein refers to the amino acidD-glutamate or D-glutamic acid.

The term "agonist" as used herein includes any compound which increasesthe flow of cations through an ionotropic receptor such as NMDA, i.e., achannel opener, and which has not been observed to decrease the flow ofcations through the same receptor.

The term "antagonist" as used herein includes any compound which reducesthe flow of cations through an ionotropic receptor such as NMDA, i.e., achannel closer, and which has not been observed to increase the flow ofcations through the same receptor.

The term "partial agonist" as used herein refers to a compound whichregulates an allosteric site on an ionotropic receptor, such as the NMDAreceptor, to increase or decrease the flux of cations through theligand-gated channel depending on the presence or absence of theprincipal site ligand. In the absence of the principal site ligand, apartial agonist increases the flow of cations through the ligand-gatedchannel, but at a lower flux than achieved by the principal site ligand.A partial agonist partially opens the receptor channel. In the presenceof the principal site ligand, a partial agonist decreases the flow ofcations through the ligand-gated channel below the flux normallyachieved by the principal site ligand. In the presence of the principalsite ligand, a partial agonist is, thus, a "partial antagonist." Theterm "principal site ligand" as used herein refers to known endogenousligands binding to a site.

The term "agonistic" as used herein refers to any compound whichincreases the flow of cations through an ionotropic receptor such asNMDA, i.e., a channel opener, and includes agonists and partialagonists.

The term "antagonistic" as used herein refers to any compound whichreduces the flow of cations through an ionotropic receptor such as NMDA,i.e., a channel closer, and includes antagonists and partial agonists.

The term "NMDA receptor" as used herein refers to a postsynapticreceptor which is stimulated, at a minimum, by the excitatory aminoacids glutamate and glycine, and selectively stimulated by the syntheticcompound NMDA. It is a ligand-gated receptor with astrychnine-insensitive glycine site.

The term "potency" as used herein refers to the molar concentration atwhich a specified effect on a receptor channel is observed.Specifically, potency for a compound exhibiting antagonistic effect ispresented as the IC₅₀ value, which is the concentration at whichinhibition of spermine-induced channel opening is 50% of the maximuminhibition achievable. Lower values indicate higher potency. Potency fora compound exhibiting agonistic effect is presented as the EC₅₀ value,which is the concentration at which enhancement of channel opening inthe absence of spermine is 50% that of maximum enhancement achievable.Again, lower values indicate higher potency.

The term "efficacious" as used herein refers to a comparison of themaximum channel opening achieved by a particular compound with themaximum channel opening achieved by spermine. Efficacy refers tomagnitude of a specified effect.

The term "Con-G site" as used herein refers to the novel complex site asdefined by the binding of Con-G, or one of its derivatives, to thepolyamine site and the novel Con site to be described below.

The term "polyamine site" as used herein refers to the site that bindsspermine and spermidine, as well as a portion of Con-G.

The term "Con site" as used herein refers to the novel site which bindsto a portion of Con-G not bound by the polyamine site.

The term "application" as used herein refers to the contacting of acompound or composition with the desired substrate either directly orindirectly, and includes in vitro, in vivo, and in situ contact. Theterm "administering" as used herein specifically refers to in vivoapplication.

The term "modulating" as used herein refers to increasing or decreasingthe flow of cations through an ionotropic receptor, such as the NMDAreceptor. "Modulators" are compounds capable of increasing and/ordecreasing the flow of cations through such receptors and includeagonists, partial agonists, and antagonists.

The term "regulating" as used herein refers to increasing or decreasingthe flow of cations through an ionotropic receptor when said flow hasbeen deviated from normal. "Regulators" are compounds capable of doingboth and, therefore, include partial agonists.

The term "neuropsychopharmacological disorder" as used herein refers toa disorder resulting from, or associated with, a reduced or excessiveflux of cations through the NMDA receptor ligand-gated cation channeland includes, but is not limited to, cognitive, learning, and memorydeficits, chemical toxicity (including substance tolerance andaddiction), excitotoxicity, neurodegenerative disorders (such asHuntington's disease, Parkinson's disease, and Alzheimer's disease),post-stroke sequelae, epilepsy, seizures, mood disorders (such asbipolar disorder, dysthymia, and seasonal effective disorder), anddepression. Neurodegenerative disorders can result from dysfunction ormalfunction of the receptor.

Design of Con G Derivatives Altering the Function of the NMDA Receptor

The discovery that Con-G modulates the response of the polyamines actingat a novel site, which includes the polyamine site and a separate Consite, and that derivatives of Con-G also modulate the effect ofpolyamines, affords an additional level of control to be exerted overthe NMDA receptor. Clearly, Con-G and its derivatives represent newclasses of antagonists, agonists, and partial agonists which modulateNMDA receptor function. Since Con-G and its derivatives modulate Ca⁺⁺influx of specific NMDA receptor sub-types, Con-G and its derivativesare expected to be similarly useful in treating the broad spectrum ofneuropsychopharmacological disorders and in enhancement of CNS functionas compounds of the prior art, but with a different specificity andspectrum of treatment.

In fact, the range of modulation of the NMDA receptor presented by Con-Gand its derivatives--from total antagonism through partial agonism tofull agonism--represents far greater control of NMDA receptor functionthan is possible with the known modulators of the Glu and Gly sites ofthe same receptor. Thus, Con-G and its derivatives possess a broaderrange of efficacies for the treatment of CNS disorders and CNS functionenhancement than is possible with compounds of the prior art. Thecompounds and methods of use are described in more detail below.

It has been postulated by others that the alignment of the charged Glaresidues is achieved through the a helix and is necessary and sufficientfor Con-G's activity at the NMDA receptor. By studying the comparativeactions of the Con-G derivatives disclosed below, it was discovered thatthe chemical/structural requirements both for the antagonistic andagonistic activities of Con-G are complex. Through preparation ofsynthetic derivatives as disclosed herein, it was determined that theability of Con-G to act as a strong, non-competitive NMDA antagonist atthe newly identified Con-G site is abolished by certain modifications ofthe Con-G peptide. In fact, it has been discovered that eithermodification of the N-terminus or replacement of the Gla residues by Gluproduces several derivatives which act as partial agonists/antagonists.

The derivatives presented in Table I below will be discussed forpurposes of illustration. It should be understood, however, that theseare representative compounds which are sufficient to teach theunderlying principles relating to structure/activity relationships topersons skilled in the art and, therefore, are not limiting of thisdisclosure.

The compounds herein described may have asymmetric centers. Unlessotherwise indicated, all chiral, diastereomeric and racemic forms areincluded. All stable isomers are contemplated. Two distinct isomers (cisand trans) of the peptide bond are known to occur; both can also bepresent in the compounds described herein. A stable isomer is one thatis sufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an useful therapeuticagent.

In Table I, the numbers above the sequences indicate each amino acid'sposition based on the parent Con-G molecule. γ-carboxyglutamate, Gla, isindicated by a lower-case "g." Where glutamate, Glu, was substituted forγ-carboxyglutamate, the single letter amino acid code for glutamate, or"E," is used. "B" refers to amino isobutyvic acid. Thenomenclature/abbreviation used to identify each compound throughout thespecification and in the claims is set out to the right beside thecorresponding polypeptide structure. The numbers in the nomenclaturerefer to the amino acid residue positions in native Con-G. In the table,and in the claims below, amino acid residues are counted from the aminoterminus to the carboxy terminus of native Con-G. Accordingly, the aminoacid at the amino terminus is designated "1." The "SEQ ID NO:" for eachpeptide is indicated in the left-most column.

In Table I, the derivatives are divided into four classes, representingthe different modifications effected. Class I represents amino terminalextensions or modifications of the native Con-G peptide. Class IIrepresents internal substitutions within the 17-amino acid peptide.Class III represents carboxy terminal fragments and modifications. ClassIV includes amino terminal fragments and modifications. The derivativesare further described below.

The experimental results obtained when testing Con-G and its derivativesfor their effect on NMDA receptor function are summarized in Table IIbelow. Select experiments are discussed in greater detail below. InTable II, the first four columns relate to determinations of ³ [H]MK-801binding in the presence of the test compound, in the absence ofspermine, indicated as "% Sti," and in the presence of the test compoundwith spermine present at maximum stimulatory concentration, indicated as"% Inh. " The concentrations at which stimulation or inhibition is 50%that of the maximum are indicated as EC (μM) and IC (μM), respectively.An "na" indicates that no activity was observed.

In Table II, those compounds exhibiting only inhibition values are fullantagonists. Those compounds exhibiting only stimulation values are fullagonists. Those compounds exhibiting inhibition and stimulation valuesare partial agonists.

As indicated in Table II, Con-G inhibits spermine-induced receptoractivity, and has no stimulatory effect on receptor activity alone.Thus, Con-G is a full antagonist. However, the derivative Con-G-OHexhibits no activity, either inhibitory or stimulatory. The Con-Gα-helix with its aligned charged Gla residues is present in Con-G-OH.Thus, an amidated carboxyl end is necessary to Con-G antagonistactivity. Further modifications of the N-terminus of Con-G also changeits behavior.

Either acylation (a mechanism known to occur naturally to stabilizepolypeptides) or the addition of the amino acid Tyr to the N-terminalresidue dramatically modifies Con-G's activity. Ac-Con-G displays noapparent activity, although the entire polypeptide sequence responsiblefor Con-G's α-helix is still present. The addition of Tyr changes Con-Gfrom a strong non-competitive antagonist of the polyamine modulatorysite to an essentially inactive compound, despite the fact that theCon-G α-helix with its aligned Gla residues remains. Thus, an unmodifiedN- terminus with a charged --NH₃ ⁺ is also necessary to Con-G antagonistactivity.

The substitution of Glu for Gla reduces the charges at each of thesubstituted sites along the side of the α-helix while preserving thehelix. This substitution also changes Con-G from a strong NMDAantagonist to a partial agonist. Substitution of all 5 Gla residues withglutamate (Glu-Con-G) gives a partial agonist, whereas substitution withD-glutamate (D-Glu-Con-G) abolished NMDA antagonist actions ofConantokin-G.

To evaluate the significance of individual Gla residues of Conantokin-Gin determining the potential contribution to biological and secondarystructure, three amino acids--alanine (Ala), serine (Ser) , andphosphoserine (Ser(p))--were substituted for Gla. Alanine can be easilyinserted into γ-helices (Chou et al., "Prediction of the secondarystructure of proteins from their amino acid sequence," Adv. Enzymol.,47:145-148, 1978; Argos, et al., "The Chou-Fasman secondary structureprediction method with an extended data base," FEBS Lett., 93:19-24,1978) but, unlike Glu and Lys, does not carry a charge and, unlike Leu,is not extremely hydrophobic. Replacement of Gla individually with Alawould not interfere with the secondary structure. Serine occupiesapproximately the same space as Ala does, but is reported to be a helixbreaker, and to help form β-turns (Chou et al., "β-turns in proteins,"J. Mol Bio., 115:135-175, 1978.) The strong, double negative charge ofGla was replaced by phosphoserine which carries a single negativecharge, but this charge is stronger than that of Glu.

The results revealed that the Gla residue in position 4 appears to berequired for the NMDA antagonist properties of the parent peptidebecause the replacement of this residue abolished NMDA antagonistactions. The substitutions of Gla in position 4 with Ala, Ser, andSer(P) resulted in a complete loss of antagonistic activity.

The Gla residue at position 3 also appears to have some effect onantagonistic properties, although not to the same extent as the Glaresidue at position 4. Ala3-Con-G inhibited 34% of binding of [³H]MK-801 produced by maximally effective concentration of spermine (12.5μM this measure) with IC₅₀ 1.8±0.1 μM (n=3). A transition fromantagonist to partial agonist activity was observed by replacing Gla inposition 3 in the order of Ala3-Con-G (antagonist) to Ser3Con-G andSer(p)3-Con-G (partial agonists).

In contrast, a Gla residue in positions 7, 10, and 14 may not benecessary for the NMDA antagonist actions. With the exception ofSer(p)7-Con-G and Ser(p)14-Con-G, the 7, 10, and 14-modified peptidesinhibited spermine-enhanced [³ H]MK-801 binding to baseline values.Indeed, Ala7-Con-G was found to be one of the most potent antagonists ofspermine with an lC₅₀ value of 45±5 nM (n=b) , approximately 4-fold(P<0.01) more potent than Con-G. Ser7-Con-G, Ser10-Con-G, Ser14-Con-G,and Ala14-Con-G displayed potencies similar to that of Con-G (Table II).Ala7-Con-G exerted its actions through a selective and noncompetitiveinhibition of spermine action, with a neurochemical profile identicalwith the parent peptide Con-G.

Strong negative charges introduced by phosphorylated serine have noconsistent effects on the biological activity of Con-G. Ser(p)7-Con-Gand Ser(p)14-Con-G partially inhibited spermine-enhanced [³ H]MK-801binding by 50 and 31% of the maximum stimulation produced by spermine,respectively, with IC₅₀ values of 1.04±0.3 (n=3) and 1.12±0.05 μM (n=3),respectively. As indicated above, Ser(p)3-Con-G exhibits partial agonistactivity. Ser(p)10-Con-G, however, exhibits full antagonist activitywith an IC₅₀ value of 0.56±0.2 μM. These observations, coupled with thefindings that the replacement of Gla residues in position 7, 10, 14 haveno major effects (although potency may be affected, as indicated by theIC₅₀ values) on the actions of Conantokin-G, suggested that thesenegative charges of Gla do not appear to be an essential element forbiological activities of Conantokin-G.

Peptide length, however, did affect observed activity. Short derivativespossessing little or no structure, but having an intact amidatedC-terminus, possess polyamine-like agonist activity. In contrast,intermediate carboxy terminal fragments and modifications exhibit noactivity. However, as the derivative length approaches the length of thenative polypeptide, an amidated C-terminus and longer structure producespartial agonist activity. The length of the structure, thus, appears tobe more important for the partial agonist function than for full agonistfunction. The effect of length in combination with helicity is discussedin more detail below.

For Con-G antagonistic activity, it appears that an amidated carboxylend, an intact charged (--NH₃ ⁺) N-terminus, a properly charged andaligned N-terminal portion, and a linker are required. Even with anamidated carboxyl end, intact N-terminus, and α-helix, modifying thecharge distribution along the whole length or the N-terminal portion (asin Glu-Con-G, Ser3-Con-G, and Ser(p)3-Con-G) changes the allostericmodulatory properties to that of a partial agomnst.

Finally, changing Con-G's N-terminus structure by lengthening Con-G, asin tBu-Tyr⁰ -Con-G and Phe⁰ -Con-G apparently interferes with the properbinding necessary for antagonism, again producing a partial agonist.Conversely, other extensions, like Tyr⁰ -Con-G, interfere with properbinding necessary for any activity. Depending on stevic interference ofthe N-terminal extension, antagonistic, agonistic, or no activityresults.

An analysis of the range of activities exhibited by the Con-Gderivatives shows that an intact amidated carboxyl terminus is requiredto achieve agonistic activity, while the N-terminus structure is crucialto achieving antagonistic or partial agonist, activities. No where isthis dichotomy more evident than in Glu-Con-G(12-17) and Con-G(14-17).Con-G(14-17), which preserves the C-terminus, is the shortestpolyamine-like agonist yet observed. It binds tightly with a potency of1.3 μM, the highest potency ever observed for a polyamine site ligand.

From the foregoing results, the general structure Formula I wasdetermined. This formula can be further divided into general structureformulas for full agonists, partial agonists, and full antagonists,respectively. For example, in one embodiment, for full agonists, x^(a)and m are zero. In another embodiment for partial agonists, m and n bothequal 1. For full antagonists m and n could either both equal 1, or ncould be zero.

Whether the activity of the Con-G derivatives as partial agonistsreflected modulation of the polyamine site directly was not clearinitially. The fact that they exhibited polyamine-like agonisticproperties suggested that they were acting at the polyamine site.Supporting this possibility is the fact that Con-G, which has beendiscovered to inhibit polyamine modulation of the polyamine site, alsoinhibits Glu-Con-G stimulated [³ H]MK-801 binding, thus suggesting thatGlu Con-G also acts at the polyamine site.

The features of Con-G and its derivatives noted above are consistentwith a physical structure of the NMDA receptor which places thepolyamine modulatory site in close proximity to the novel Con site sothat, together, they form the novel Con-G complex site (i.e., the "Con-Gsite"). As with all protein structures, although the sites may well bein close physical proximity, they may be separated by many interveningresidues along the folded protein.

As depicted in FIG. 2, the N-terminus of Con-G binds to one part of theCon-G site (designated the "Con site") while the C-terminus binds to atleast a portion of the polyamine site. A certain length appearsnecessary to bridge the distance between the two sites, as evidenced bythe results obtained with the derivatives. At least some of the highlycharged Gla residues, in particular at amino acid position four,apparently are responsible for binding to the correct and criticaldeterminants of the NMDA receptor for antagonistic function.

The action of Con-G, thus, is similar to that of other known ligandswhich bind in such a manner as to effect a relative movement ofadjoining protein segments towards or away from each other, to cause theopening and closing of an ion channel. Given this understanding of thestructural requirements for Con-G activity, it is possible to designmolecular analogs of Con-G and its derivatives which possess therequired binding characteristics, and which are modified to increase ordecrease the modulatory effects (activities) at these sites. Thus, notonly has the mode of action of Con-G been elucidated, but also a newclass of compounds has been discovered which allosterically modulate apolyamine associated site of the NMDA receptor over a very wide range ofactivities.

As indicated above, new classes of allosteric modulators of the Con-Gsite, acting at the polyamine site and/or the Con G site, have beendiscovered which exhibit a range of activities from full antagonism, topartial agonism, to full agonism. This class of compounds may be used tomodulate the flow of ions through the NMDA receptor. The range ofmodulation of the compounds acting at the site goes from partiallyinhibitory (down modulatory) to partially stimulatory (up modulatory).Depending on the required activity, compounds from this class can beused as pharmaceutical neuroprotectants to treat acute cases of massiveCa⁺⁺ influx due to CNS injury and trauma, as well as to treatconvulsions, mood disorders, and other neuropsychiatric andneurodegenerative diseases due to chronic disturbances in control ofCa⁺⁺ influx. Similarly, compounds of this class can be selected for therequired activity to treat cognitive deficits and to enhance memory andlearning.

The discovery that Con-G acts as a non-competitive antagonist ofpolyamine stimulation of the NMDA receptor suggests that it can be usedpharmaceutically to modulate/regulate the NMDA receptor response both inthose instances where the enhanced Ca⁺⁺ flux through the NMDA receptoris stimulated by polyamines and in those situations where excessstimulation by other allosteric modulators relies on an appropriatelevel of polyamines being present and active.

Pharmaceutical Compositions

The compounds can be administered parenterally, i.e. subcutaneously,intramuscularly, intracerebroventricularly, or intravenously and,alternatively, intrathecally. In appropriate carriers or in combinationwith agents enhancing passage of the blood brain barrier, they can beadministered orally or nasally.

Suitable pharmaceutical carriers are known to those skilled in the art.For example, when the active ingredient is administered parenterally, insterile liquid dosage forms, the carrier can be water, a suitable oil,saline or other buffered physiological solution, aqueous dextrose orrelated sugar solutions and glycols, such as propylene glycol orpolyethylene glycol. Solutions for parenteral administration preferablycontain a water soluble form of the active ingredient, suitablestabilizing agents, and if necessary, buffer substances. Antioxidizingagents such as sodium bisulfite, sodium sulfite, or ascorbic acid eitheralone or combined are suitable stabilizing agents. Also used are citricacid and its salts and sodium EDTA. In addition, parenteral solutionscan contain preservatives, such as benzalkonium chloride, methyl- orpropylparaben, and chlorobutanol. Suitable pharmaceutical carriers canbe included and are described in Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Co., Easton, Pa, p.1418 (1985), a standardreference text in this field incorporated herein by reference).

The active ingredient can be administered orally in solid dosage forms,such as capsules, tablets and powders, or in liquid dosage forms, suchas elixirs, syrups, and suspensions, if first encapsulated in an agentwhich is stable to passage through the gastrointestinal tract and whichallow passage through the blood brain barrier, such as some of thestabilized or covalently crosslinked liposomes. Passage through theblood brain barrier can be enhanced using agents such as some of thephospholipids or lecithin derivatives described in the literature.

Other agents that can be used for delivery include liposomes,microparticles (including microspheres and microcapsules), and otherrelease devices and forms that provide controlled, prolonged or pulsed,delivery or which enhance passage through the blood brain barrier, forexample. These are most preferably implanted at or within the bloodbrain barrier to provide controlled release over an appropriate periodof time.

Bioerodible microspheres can be prepared using any of the methodsdeveloped for making microspheres for drug delivery, for example, asdescribed by Mathiowitz and Langer, J. Controlled Release 5,13-22(1987); Mathiowitz, et al., Reactive Polymers 6, 275-283 (1987); andMathiowitz, et al., J. Appl. Polymer Sci. 35, 755-774 (1988), theteachings of which are incorporated herein. The selection of the methoddepends on the polymer selection, the size, external morphology, andcrystallinity that is desired, as described, for example, by Mathiowitz,et al., Scanning Microscopy 4,329-340 (1990); Mathiowitz, et al., J.Appl. Polymer Sci. 45, 125-134 (1992); and Benita, et al., J. Pharm.Sci. 73, 1721-1724 (1984), the teachings of which are incorporatedherein. Methods routinely used by those skilled in the art includesolvent evaporation, hot melt encapsulation, solvent removal, spraydrying, phase separation and ionic crosslinking of gel-type polymerssuch as alginate or polyphosphazines or other dicarboxylic polymers toform hydrogels.

Other delivery systems including films, coatings, pellets, slabs, anddevices can be fabricated using solvent or melt casting, and extrusion,as well as standard methods for making composites.

The microparticles can be suspended in any appropriate pharmaceuticalcarrier, such as saline, for administration to a patient. In the mostpreferred embodiment, the microparticles will be stored in dry orlyophilized form until immediately before administration. They will thenbe suspended in sufficient solution for administration. The polymericmicroparticles can be administered by injection, infusion, implantation,orally, or administration to a mucosal surface, for example, thenasal-pharyngeal region and/or lungs using an aerosol. The other devicesare preferably administered by implantation in the area where release isdesired. Lower dosages are used with implantable controlled releasedevices than with other forms of administration other than directadministration to the brain.

The appropriate dosages will depend upon the route of administration andthe treatment indicated, and can be readily determined by one skilled inthe art. Dosages are generally initiated at lower levels and increaseduntil desired effects are achieved. A dosage range of 1 to 40 mg/kg bodyweight is contemplated. These dosages are based on extrapolation fromstudies using compounds of similar size peptides, such as WO 93/10145,originally filed Nov. 12, 1991, which describes compositions for thedelayed treatment of ischemia-related neuronal damage, including theomega conotoxin peptide OCT MVIIA, which is 25 amino acids long.Neuroprotective effects were reported with intravenous administration ofOCT MVIIA at doses of 15 mg/kg and less. U.S. Pat. No. 5,051,403, issuedSep. 24, 1991, described intracerebroventricularly injection of omegaconotoxin peptides to reduce anatomical damage resulting from globalischemia. Doses of 1 μg or less per 50-80 gram gerbil were used.Protective effects were observed at dosages of less than 0.1 μg.

Assays for Determining Functional Activity of Compounds

The following methods and materials were used to determine the activityof the peptides described herein. They can be used with only routineexperimentation to test many other peptide embodiments derived from ConG for use as partial agonists, agonists, or antagonists, as describedherein.

Peptide Synthesis

Con-G was synthesized and purified by a modification of the method ofRivier et al., Biochemistry, 26:8508-8512 (1987), incorporated herein byreference. Amino acid derivatives and solid-phase resins were obtainedfrom Bachem Feinchemikalien AG (Bubendorf, Switzerland) and Novabiochem.In general, peptides were synthesized on a MilliGen 9050 automatedpeptide synthesizer by using a polystyrene-polyepoxy graft copolymerresin (Rapp et al., Innovation and Perspectives in Solid PhaseSynthesis, R. Epton, ed., SPCC, Birmingham, pp 205-210, 1990,incorporated herein by reference). Standard Fmoc-chemistry(9-fluorenylmethyloxycarbonyl, Fields et al., "Solid-phase peptidesynthesis utilizing 9-fluorenylmethoxycarbonyl amino acids," Int. J.Peptide Protein Res., 35:161-214, 1990, incorporated herein byreference) was used throughout. The amino acids were coupled with a4-fold molar excess of pentafluorophenyl esters, HBTU or TETU. Peptideswere cleaved from the solid support with trifluoroacetic acid (TFA)containing 5% of thioanisole as scavenger. The peptides wereprecipitated with ether and purified with reversed-phase highperformance liquid chromatography (RP-HPLC) . A minor fragment of thepeptides was purified in the absence of TFA, to exclude the presence oflarge amounts of counter ions that may interfere with the conformationalanalysis. The integrity of the peptides was verified by amino acidanalysis and fast atom bombardment mass spectroscopy.

Fmoc-Gla (di-t-Bu-OH) was coupled with smaller molar excess (2 to 2.5)with a coupling time of 90 minutes. Synthesis of Tyr⁰ -Con-G wasaccomplished by an additional carbodiimide-mediated coupling step withFmoc-Tyr (O-tBu-OH) to the final peptide resin. The N.sup.α -acetylCon-G (Ac-Con-G) analog was prepared by treating the peptide resin with20% piperidine in dimethylformamide (DMF) for 30 minutes to remove theFmoc group. Following a series of washes with DMF, dichloromethane, andisopropyl alcohol, a positive Kaiser test (Kaiser et al., Analyt.Biochem., 34:595, 1970, incorporated herein by reference) was obtainedindicating complete removal of the Fmoc group. The peptide-resin wastreated with 20% acetic anhydride in DMF for 30 minutes. A negativeKaiser test indicated complete blocking of the N-terminal amino group.

All analogs not containing Gla were synthesized using a BOC/Bzl(t-butyloxycarbonyl/benzyl) strategy incorporating the preprogranmmedprotocols of an automated peptide synthesizer (Applied Biosystems 431-A,Foster City, Calif. Cleavage and deprotection of each of the analogs wasaccomplished by treatment with liquid hydrogen fluoride (HF) in thepresence of m-cresol. Each analog was purified to homogeneity byreverse-phase HPLC on a Waters C₁₈ silicon column using a lineargradient from 10% to 30% acetonitrile into H₂ O containing 0.1 % TFAover 60 minutes. Highly pure fractions were combined and lyophilized.Amino acid analysis results were within 10% of theoretical for eachpeptide.

In the synthesis of phosphopeptides, the serine residue to bephosphorylated was incorporated with its side-chain hydroxyl groupunprotected, and phosphorylation was carried out on the resin after thepeptide assembly was completed by using dibenzyl phosphochloridate(Otvos et al., "Solid-phase synthesis of phosphopeptides," Int. J.Peptide Protein Res., 34:129-133, 1989, incorporated herein byreference). The phosphopeptides were purified by RP-HPLC (Otvos et al.,"Reversed phase high performance liquid chromatographic separation ofphosphopeptide isomers," J. Chromatography, 512:265-272 (1990),incorporated herein by reference) and analyzed by phosphoaminoacid-sensitive amino acid analysis (Gorbics et al., "Successful andrapid verification of the presence of a phosphate group in syntheticphosphopeptides using the conditions of standard dabs-Cl amino acidanalysis," J. Liquid Chromatogr., 17:175-189, 1994, incorporated hereinby reference) and mass spectroscopy. This synthetic strategy providedserine and phosphoserine-containing peptides at the same time.

Binding Assays

Membrane Preparation:

Male Sprague-Dawley rats (175-300 grams, Taconic Farms, Germantown,N.Y.) were killed by decapitation. Forebrains minus cerebellum and brainstem were removed and homogenized with a Polytron homogenizer (setting6, 30 seconds) using 10 volumes of 0.32 M sucrose in the assay buffer (5mM Hepes/4.5 Mm Tris buffer, Ph 7.8). All procedures were carried out at4° C., unless specified otherwise. The homogenate was diluted to 50volumes with assay buffer and centrifuged at 1,000×g for 10 minutes. Thesupernatant was decanted and recentrifuged at 20,000×g for 20 minutes.The resulting pellet was resuspended in 50 volumes of assay buffer andcentrifuged at 8,000×g for 20 minutes. The supernatant and outer "buffy"pellet coat were collected and centrifuged at 20,000×g for 20 minutes.The supernatant was discarded, and the pellet was resuspended in 50volumes of assay buffer containing 1 mM EDTA prior to recentrifugationat 20,000×g for 20 minutes. This resuspension/centrifugation procedurewas repeated 3-4 times. The last cycle(s) were performed using assaybuffer without EDTA. The resulting pellet was resuspended in 5 volumesof assay buffer, frozen over solid CO₂, and stored at -70° C. On the dayof assay the tissue was thawed, diluted with 10-50 volumes of assaybuffer, and centrifuged at 20,000×g for 20 minutes. The supernatant wasdiscarded, and the resulting pellet was resuspended in 50 volumes ofassay buffer and centrifuged at 20,000×g for 20 minutes. The finalpellet was resuspended in 30-50 volumes of assay buffer without furthermodification.

Radioligand Binding

Binding assays were performed in a total volume of 500 μl containingapproximately 40-200 μl containing approximately 40-200 μg protein(membrane preparation), 50 μl [³ H]MK-801 (final concentration, 4-5 nM),and test compounds or buffer. [³ H]MK-801 (specific activity 28.8Ci/mmol) was obtained from DuPont-NEN (Boston, Mass.). Assays wereincubated for 2 hours at room temperature and terminated by rapidfiltration under partial vacuum (Brandel Cell Harvester, Model M-24R)with two 5 ml washes of buffer over glass fiber filters that werepresoaked in 0.03% polyethyleneimine. Nonspecific binding was determinedusing phencyclidine hydrochloride (PCP, 100 μM) and represented about15-50% of the total binding in the absence of modulatory agents.Radioactivity retained in the filter was measured in Ultima Goldscintillation liquid using a Packard 1600TR liquid scintillationcounter. Protein content was determined using the BCA Protein AssayReagent (Pierce, Rockford, Ill.).

The following assays can be used to study in vivo activity.

Mongolian gerbil Forebrain Ischemia Assay

The Mongolian gerbil forebrain ischemia assay is used to determine theextent of protection afforded by a test compound on neural brain cellssubjected to ischemic conditions as a model of neurodegeneration. MaleMongolian gerbils are injected ip, iv, and icv with the test compoundprior to carotid occlusion. Carotid flow is then occluded for 5-20minutes by clamps and then opened and inspected to confirm reflow. Thegerbils are kept alive for 7 days following surgery and thenanesthetized with pentobarbital and perfused transcardially with salinewith heparin followed by buffered formalin. The brain is removed,trimmed, and prepared for histological processing. Brain sections arestained and damaged neurons in the CAl region of the hippocampus areexamined. The effects of the test compound are compared to untreatedcontrols. Cell loss is reduced in the gerbils pretreated with testcompounds exerting a protective effect against ischemia-inducedneurodegeneration. The compounds are expected to be active in this testat a dose of about 1-40 mg/kg, iv or ip.

Forced Swim Test

Compounds with antidepressant activity reduce the time of mouseimmobility as measured by the "forced swim test" described by Trullos etal., "Functional antagonists at the NMDA receptor complex exhibitantidepressant actions," Eur. J. Phann., 185:1-10 (1990) and referencestherein (incorporated herein by reference). Mice are placed individuallyin a cylinder (i.e., having a diameter of 10 cm and height of 25 cm)filled with water (6 cm) at 22-25° C. The duration of immobility isscored during the last four minutes of a six minute test. The compoundsare expected to be active in this test at a dose of about 1-40 mg/kg ip.

Elevated Plus Maze

Compounds with antidepressant activity increase both the percentage oftime and percentage of entries into the open arms of an elevatedplus-maze as described by Trullos et al.,"1-Aminocyclopropanecarboxylates exhibit antidepressant and anxiolyticactions in animal models," Eur. J. Pharm., 203:379-385 (1991),incorporated herein by reference. A mouse is placed at the intersectionof the maze arms so that its head is in the center of the platform. Themouse is then scored as being in the open or enclosed arms. Arm entriesare recorded and the percentage of time in each arm, as well as thepercentage of entries, are calculated. The compounds are expected to beactive in this test at a dose of about 1-40 mg/kg ip.

NMDA Induced Seizures

Compounds which have anticonvulsant activity for convulsions involvingthe NMDA receptor are active in a test described by Koek et al.,Mechanisms for Neuromodulation and Neuroprotection, pp 665-671, Kamenkaet al., eds., NPP Books, Ann Arbor, Mich., 1992, incorporated herein byreference. Test compounds are injected into mice at 15 minutes or 30minutes before an ip injection of NMDA, icv or ip, respectively. ED⁵⁰ isdetermined by comparing the percentage of mice that die after 30 minutesto a group of mice that receive NMDA alone. The compounds are expectedto be active in this test at a dose of about 1-40 μg/kg icv, or 1-40mg/kg ip.

Cocaine Induced Convulsions

Compounds with anticonvulsant activity for cocaine induced convulsionsare active in tests described by Witkin et al., Life Sciences, 48:51-56,1991, incorporated herein by reference. Male Swiss Webster mice, 10-12weeks old, are injected with the test compound ip 30 minutes prior to anip injection of 75 mg/kg cocaine. The occurrence of convulsions isrecorded for 15 minutes following the cocaine injection and are definedas loss of righting responses for at least 5 seconds and the occurrenceof clonic limb movement. The ED₅₀ dose can then be calculated. Thecompounds are expected to be active in this test at doses of about 1-40mg/mg ip.

EXAMPLE 1 Characterization of Con G Activity

FIGS. 3a and 3b are plots of the binding of [³ H]MK-801 as a function ofincreasing concentrations of spermine and spermidine, respectively, inthe nominal absence of endogenous Glu and Gly. While the forebrainmembrane preparations were washed very well, as noted in the methodssection above, it is believed that it is not entirely possible tototally remove these amino acids from the membrane preparations, asindicated by the high non-specific binding in such assays. As can beseen from the figures, both of these polyamines stimulate the binding of[³ H]MK-801 in a concentration dependent fashion, confirming that theyallosterically modulate the opening of the ligand-gated NMDA channel.

FIGS. 4a and 4b depict the effect on spennine- and spermidine-stimulatedbinding of [³ H]MK-801, as depicted in FIGS. 3a and 3b, when varyingamounts of Con-G are added to the membrane preparation. In the figures,squares represent no added Con-G; triangles represent Con-G at 400 μM;diamonds represent Con-G at 800 μM; and circles represent Con-G at 1200μM.

As can be seen from the figures, as the concentration of Con-G increasesfrom 400 μM to 1200 μM, the degree of polyamine stimulated [³ H]MK-801binding concomitantly decreases, thereby reflecting a more and moreclosed state of the channel. Con-G does not decrease the polyaminestimulated binding in a concentration dependent manner which would beconsistent with competitive inhibition, but does decrease thestimulation in a manner consistent with non-competitive inhibition ofthe polyamines.

FIG. 5 is a plot of the reduction in maximum stimulation of [³ H]MK-801binding achieved by spermine (squares) and spermidine (triangles), at 50μM concentration each, as the concentration of Con-G increases. There isno consistent relationship between the degree of inhibition of thepolyamine stimulation and the concentration of Con-G such as is foundwith the polyamines diethylenetriamine and arcaine, both of whichinhibit spermine and spermidine enhanced [³ H]MK-801 binding through acompetitive antagonism at the polyamine site, further indicating thatCon-G is not acting as a competitive inhibitor of the polyamines at thepolyamine site.

In FIG. 13, the effect of increasing concentrations of Con-G upon basal[³ H]MK-801 binding is depicted. The curves preceding the break (├┤)represent [³ H]MK-801 binding in the absence of Con-G. As can be seenfrom FIG. 13, Con-G produces a small increase in basal [³ H]MK-801binding. This increase is very modest compared to the enhanced bindingproduced by Glu, Gly, and the polyamines.

EXAMPLE 2 Con G Derivatives Exhibiting Full Antagonist Activity.

The peptides disclosed in Table I were tested for their effect onspermine-induced binding of [³ H]MK-801 in the radioligand binding assaydescribed previously. All the compounds with antagonistic activities areactive in this assay.

As shown in FIG. 4, Con-G acts as a very strong antagonist. Like nativepeptide Con-G, Ala7-Con-G inhibited spermine-induced binding, and in anon-competitive manner. This was manifested by analysis ofconcentration--response data of spermine generated in the presence ofvarious concentrations of Ala7-Con-G. There was a progressivedownshifting of concentration-response curves of spermine as theconcentrations of Ala7 increased. Results are depicted in FIG. 6.

In FIG. 6, concentration-response curves were obtained in the presenceof 12.5 (opened symbols) and 25 (filled symbols) μM spermine,respectively. The symbols in FIG. 6 are as follows: open circles,Ala7-Con-G; open diamonds, Con-G; and dotted line, spermine alone. Theresults shown are from a typical experiment performed in duplicate, inwhich IC₅₀ values of Ala7-Con- G and Con-G were 50 and 275 nM,respectively. Moreover, the apparent IC₅₀. of Ala7-Con-G was unaffectedby spermine concentrations. Both features were consistent withnon-competitive antagonism.

At 10 nM, Ala7-Con-G inhibited about 50% of the maximum stimulationproduced by spermine, and the concentration-response curve of sperminewas reduced almost to baseline values by a concentration of 30 nMAla7-Con-G. Results are depicted in FIG. 7. Non-competitive inhibitionof polyamine-enhanced [³ H]MK-801 binding by Ala7 is shown.Spermine-enhanced [³ H]MK-801 binding was determined in the presence of0 (squares), 10 (diamonds), and 30 nM Ala7-Con-G (triangles),respectively. The results shown were from a typical experiment induplicate and were repeated with similar results.

                                      TABLE I                                     __________________________________________________________________________    Con G Peptides                                                                SEQ ID NO.:                                                                           SEQUENCE       DESIGNATION                                            __________________________________________________________________________    SEQ ID NO: 1                                                                          GEggLQgNQgLIRgKSN                                                                            ConantokinG                                            Class I - Amino Terminal Extensions/Modifications                             SEQ ID No: 4                                                                          Ac-GEggLQgNQgLIRgKSN                                                                         Ac-Con-G                                                 SEQ ID No: 13 AcY-GEggLQgNQgLIRgKSN Ac-Tyr.sup.o -Con-G                       SEQ ID No: 14 N-GEggLQgNQgLIRgKSN Asn.sup.o -Con-G                            SEQ ID No: 15 N(GIc0c)-GEggLQgNQgLIRgKSN Asn(GlcN Ac).sup.o -Con-G                                  SEQ ID No: 16 F-GEggLQgNQgLIRgKSN Phe.sup.o                                  -Con-G                                                   SEQ ID No: 17 F(pCl)-GEggLQgNQgLIRgKSN Phe(pCl).sup.o -Con-G                  SEQ ID No: 18 Y(p)-GEggLQgNQgLIRgKSN Tyr(p).sup.o -Con-G                      SEQ ID No: 19 Y(oMe)-GEggLQgNQgLIRgKSN Tyr(oMe).sup.o -Con-G                  SEQ ID No: 3 Y-GEggLQgNQgLIRgKSN Tyr.sup.o -Con-G                             SEQ ID No: 20 tBu-Y-GEggLQgNQgLIRgKSN tBu-Tyr.sup.o -Con-G                    SEQ ID No: 21 S-GEggLQgNQgLIRgKSN Ser.sup.o -Con-G                          Class II - Internal Substitutions                                             SEQ ID No: 5                                                                          GEEELQENQELIREKSN                                                                            Glu3,4,7,10,14-Con-G                                                                      (Glu-Con-G)                                  SEQ ID No: 11 GdEdEdELQdENQdELIRdEKSN D-Glu3,4,7,10,14-Con-G (D-Glu-Con-                                       G)                                           SEQ ID No: 6 Y-GEEELQENQELIREKSN Tyr.sup.o -Glu3,4,7,10,14-Con-G                                                SEQ ID No: 22 GEAgLQgNQgLIRgKSN                                              Ala3-Con-G                                   SEQ ID No: 23 GESgLQgNQgLIRgKSN Ser3-Con-G                                    SEQ ID No: 24 GES(p)gLQgNQgLIRgKSN Ser(p)3-Con-G                              SEQ ID No: 25 GEYgLQgNQgLIRgKSN Tyr3-Con-G                                    SEQ ID No: 26 GEgELQgNQgLIRgKSN Glu4-Con-G                                    SEQ ID No: 27 GEgALQgNQgLIRgKSN Ala4-Con-G                                    SEQ ID No: 28 GEgSLQgNQgLIRgKSN Ser4-Con-G                                    SEQ ID No: 29 GEgS(p)LQgNQgLIRgKSN Ser(p)4-Con-G                              SEQ ID No: 30 GEggYQgNQgLIRgKSN Tyr5-Con-G                                    SEQ ID No: 31 GEggLQYNQgLIRgKSN Tyr7-Con-G                                    SEQ ID No: 53 GEggLQY(I)NQgLIRgKSN iodo-Tyr7-Con-G                            SEQ ID No: 54 GEggLQY(II)NQgLIRgKSN di-iodo-Tyr7-Con-G                        SEQ ID No: 32 GEggLQANQgLIRgKSN Ala7-Con-G                                    SEQ ID No: 33 GEggLQSNQgLIRgKSN Ser7-Con-G                                    SEQ ID No: 34 GEggLQS(p)NQgLIRgKSN Ser(p)7-Con-G                              SEQ ID No: 35 GEggLQgNQALIRgKSN Ala10-Con-G                                   SEQ ID No: 36 GEggLQgNQSLIRgKSN Ser10-Con-G                                   SEQ ID No: 37 GEggLQgNQS(p)LIRgKSN Ser(p)10-Con-G                             SEQ ID No: 38 GEggLQgNQgLIRAKSN Ala 14-Con-G                                  SEQ ID No: 39 GEggLQgNQgLIRSKSN Ser14-Con-G                                   SEQ ID No: 40 GEggLQgNQgLIRS(p)KSN Ser(p) 14-Con-G                            SEQ ID No: 41 GEggLQENQELIREKSN Glu,7,10,14-Con-G                             SEQ ID No: 42 GEggLQANQALIRAKSN Ala 7,10,14-Con-G                             SEQ ID No: 55 GEggLQSNVSQIRAKSN 3.sub.10  helix ConG                          SEQ ID No: 56 GEggLQAALALIRAKSN alpha helix ConG                              SEQ ID No: 65: GEggL-cyclic     cyclic ConG(1-5)                              SEQ ID No: 57 GEggL-gKSN ConG(1-5)(14-17)                                     SEQ ID No: 58 GEggL-GG-gKSN ConG(1-5)GG(14-17)                                SEQ ID No: 59 GEggL-GGGG-gKSN ConG(1-5)GGGG(14-17)                            SEQ ID No: 60 GEggL-AA-gKSN ConG(1-5)AA(14-17)                                SEQ ID No: 61 GEggL-AAA-gKSN ConG(1-5)AAAA(14-17)                             SEQ ID No: 62 GEggLQ-AAAAA-gKSN ConG(1-6)A5(14-17)                            SEQ ID No: 63 GEggLQ-AAAAAAA-gKSN ConG(1-6)A7(14-17)                          SEQ ID No: 64 GEggL-BB-gKSN ConG(1-5)BB(14-17)                              Class III - Carboxy Terminal Fragments/Modifications                          SEQ ID No: 7                                                                          IREKSN         Glu 14-Con-G(12-17)                                      SEQ ID No: 43 IREASN Glu 14-Ala 15-Con-G(12-17)                               SEQ ID No: 44 IAEKSN Ala 13, Glu 14-Con-G(12-17)                              SEQ ID No: 45 IAEASN Ala 13, 15, Glu 14-Con-G(12-17)                          SEQ ID No: 8 QELIREKSN Glu 10,14-Con-G(9-17)                                  SEQ ID No: 9 QENQELIREKSN Glu 7,10,14-Con-G(6-17)                             SEQ ID No: 10 EEELQENQELIREKSN Glu 3,4,7,10,14-Con-G(2-17)                    SEQ ID No: 46 IRgKSN Con-G(12-17)                                             SEQ ID No: 47 RSgNK scramblcd Con-G(13-17)                                    SEQ ID No: 48 RgKSN Con-G(13-17)                                              SEQ ID No: 49 gKSN Con-G(14-17)                                               SEQ ID No: 50 IRgK Con-G(12-15)                                               SEQ ID No: 51 KSN Con-G(15-17)                                              Class IV - Amino Terminal Fragments/Modifications                             SEQ ID No: 2                                                                          GEggLQgNQgLIRgKSN-OH                                                                         Con-G-OH                                                 SEQ ID No: 52 GEggL Con-G(1-5)                                              __________________________________________________________________________

                                      TABLE II                                    __________________________________________________________________________                               Spermine   %                                         Structure EC (μM) % Sti IC (μM) Inh                                   __________________________________________________________________________    Spermine              4.5  100  0     0                                         Spermidine 10.8 97 0 0                                                        Mg.sup.2+ 20.6 52 0 0                                                         ConantokinG 0 0 0.16 100                                                      SEQ ID No: 1                                                                Class I - Amino Terminal Extensions/Modifications                             SEQ ID NO: 4                                                                          Ac-Con-G      0    0    0     0                                         SEQ ID NO: 13 Ac-Tyr.sup.o -Con-G 0 0 18.3 73                                 SEQ ID NO: 14 Asn.sup.o -Con-G 0 0 8.7 83                                     SEQ ID NO: 15 Asn(GlcNAc).sup.o -Con-G 0 0 8.8 38                             SEQ ID NO: 16 Phe.sup.o -Con-G 6.8 ± 1.6 53 ± 17  6.1 51                SEQ ID NO: 17 Phe(pCl).sup.o -Con-G 0 0 0 0                                   SEQ ID NO: 18 Tyr(p).sup.o -Con-G 0 0 0 0                                     SEQ ID NO: 19 Tyr(oMe).sup.o -Con-G 0 0 0 0                                   SEQ ID NO: 3 Tyr.sup.o -Con-G 0 0 0 0                                         SEQ ID NO: 20 tBu-Tyr.sup.o -Con-G 1.0 69 1.5 29                              SEQ ID NO: 21 Ser.sup.o -Con-G 0 0 6.8 41                                   Class II- Internal Substitutions                                              SEQ ID NO: 5                                                                          Glu3,4,7,10,14-Con-G                                                                        6.7  79   8.5   22                                         (Glu-Con-G)                                                                  SEQ ID NO: 11 D-Glu3,4,7,10,14-Con-G 5.8 30 0 0                                (D-Glu-Con-G)                                                                SEQ ID NO: 6 Tyr.sup.o -Glu3,4,7,10,14-Con-G 2.7 31 0 0                       SEQ ID NO: 22 Ala3-Con-G 0 0 1.8 ± 0.1 34 ± 9                           SEQ ID NO: 23 Ser3-Con-G 8.4 ± 1.6 62 ± 4.9 3.6 ± 0.7 23 ±                                                 3.6                                  SEQ ID NO: 24 Ser(p)3-Con-G 3.5 ± 1.9 68 ± 8.4  0.9 ± 0.09 34                                                 ± 2.3                             SEQ ID NO: 25 Tyr3-Con-G 0 0 0 0                                              SEQ ID NO: 26 Glu4-Con-G 0 0 0 0                                              SEQ ID NO: 27 Ala4-Con-G 0 0 0 0                                              SEQ ID NO: 28 Ser4-Con-G 0 0 0 0                                              SEQ ID NO: 29 Ser(p)4-Con-G 0 0 0 0                                           SEQ ID NO: 31 Tyr7-Con-G 0 0 0.5 100                                          SEQ ID NO: 53 iodo-Tyr7-Con-G 0 0 0.5 100                                     SEQ ID NO: 54 di-iodo-Tyr7-Con-G 0 0 0.5 100                                  SEQ ID NO: 32 Ala7-Con-G 0 0 0.046 100                                        SEQ ID NO: 33 Ser7-Con-G 0 0 0.25 ± 0.04 100                               SEQ ID NO: 34 Ser(p)7-Con-G 0 0 1.04 ± 0.3  50 ± 3.8                    SEQ ID NO: 35 Ala10-Con-G 0 0 1.10 ± 0.3  100                              SEQ ID NO: 36 Ser10-Con-G 0 0 1.80 ± 0.2  100                              SEQ ID NO: 37 Ser(p)10-Con-G 0 0 0.56 ± 0.2  100                           SEQ ID NO: 38 Ala14-Con-G 0 0 0.16 ± 0.03 100                              SEQ ID NO: 39 Ser14-Con-G 0 0 0.16 ± 0.04 100                              SEQ ID NO: 40 Ser(p)14-Con-G 0 0 1.12 ± 0.05 31 ± 0.5                   SEQ ID NO: 41 Glu7,10,14-Con-G 0 0 >5 70 4                                    SEQ ID NO: 42 Ala7,10,14-Con-G 0 0 0.5 100 4                                  SEQ ID No: 55 3.sub.10  helix ConG 0 0 20 25 3                                SEQ ID No: 56 alpha helix ConG 0 0 1.5 100 5                                  SEQ ID No: 57 ConG(1-5)(14-17) 0 0 100 15 2                                   SEQ ID No: 58 ConG(1-5)GG(14-17) 0 0   1                                      SEQ ID No: 59 ConG(1-5)GGGG(14-17) 0 0   1                                    SEQ ID No: 60 ConG(1-5)AA(14-17) 0 0   3                                      SEQ ID No: 61 ConG(1-5)AAAA(14-17) 0 0   3                                    SEQ ID No: 62 ConG(1-6)A5(14-17) 0 0   3-4                                    SEQ ID No: 63 ConG(1-6)A7(14-17) 0 0   4-5                                    SEQ ID No: 64 ConG(1-5)BB(14-17) 0 0 15 25 2-3                              Class III - Carboxy Terminal Fragments/Modifications                          SEQ ID NO: 7                                                                          Glu14-Con-G(12-17)                                                                          47   30   0     0                                         SEQ ID NO: 43 Glu 14, Ala15-Con-G(12-17)   inactive                           SEQ ID NO: 44 Ala13, Glu14-Con-G(12-17)   inactive                            SEQ ID NO: 45 Ala13,15, Glu14-Con-G(12-17)   inactive                         SEQ ID NO: 8 Glu10,14-Con-G(9-17) 0 0 0 0                                     SEQ ID NO: 9 Glu 7,10,14-Con-G (6-17) 0 0 0 0                                 SEQ ID NO: 10 Glu3,4,7,10-Con-G(2-17) 0.9 33 5.4 70                           SEQ ID NO: 46 Con-G(12-17) 58 20 0 0                                          SEQ ID NO: 47 scrambled Con-G   inactive                                      SEQ ID NO: 48 Con-G(13-17)   inactive                                         SEQ ID NO: 49 Con-G(14-17) 1.3 ± 0.6 61 ± 1.3 0 0                       SEQ ID NO: 50 Con-G(12-15) 69.75 ˜20 0 0                                SEQ ID NO: 51 Con-G(15-17) 61.72 ˜20 0 0                              Class IV - Amino Terminal Fragments/Modifications                             SEQ ID NO: 2                                                                          Con-G-OH      0    0    0     0                                         SEQ ID NO: 52 Con-G(1-5) 0 0 7.43 50                                          SEQ ID NO: 65 Cyclic ConG(1-5)   inactive                                   __________________________________________________________________________

EXAMPLE 3 Con G Derivatives Exhibiting Partial Agonist Activity.

As indicated in Table II, several of the derivatives exhibited partialagonist activity. The results for two of these, tBu-Tyr⁰ -Con-G andGlu-Con-G, are depicted in FIG. 8. In FIG. 8, the dotted line representsthe level of binding achieved with a maximally effective concentrationof spermine, i.e., 25 μM. The top two curves depict that, at higherconcentrations, Glu-Con-G (diamonds) and tBu-Tyr⁰ -Con-G (circles) areantagonistic to spermine induced enhancement of [³ H]MK-801 binding.Although Glu-Con-G and tBu-Tyr⁰ -Con-G exhibit antagonism (top curves)to polyamine enhanced [³ H]MK-801 binding at higher concentrations, thelevels of such antagonism are nowhere near those characteristic of thenative Con-G. In fact, a concentration of tBu-Tyr⁰ -Con-G one order ofmagnitude greater than that of Con-G is required to obtain the samedecrease as Con-G. The concentration of Glu-Con-G required is one orderof magnitude greater than that of tBu-Tyr⁰ -Con-G. The bottom two curvesof FIG. 8 depict that, in the absence of spermine, both Glu-Con-G(triangles) and tBu-Tyr⁰ -Con-G (squares) are agonists which enhance [³H]MK-801 binding in a concentration-dependent manner.

In addition to Glu-Con-G and tBu-Tyr⁰ -Con-G, several other derivativesof Con-G show similar concentration dependent enhancements of [³H]MK-801 binding in the absence of polyamines. Ser3-Con-G andSer(p)3-Con-G inhibited spermine-enhanced (12.5 μM) [³ H]MK-801 bindingto 73 and 65% of the maximum values, with IC₅₀ values of 3.6±0.7 (n=3)and 0.9±0.09 μM (n=3), respectively. This is depicted in FIG. 9:Ser3-Con-G, filled diamonds; Ser(p)3-Con-G, filled circles. In theabsence of spermine, Ser3-Con-G and Ser(p)3-Con-G stimulation of [³H]MK-801 binding (open diamonds and open circles, respectively).Ser3-Con-G and Ser(p)3-Con-G increased [³ H]MK-801 binding withpotencies similar to that of spermine, but significantly lessefficacious. Glu-Con-G(2-17) and Phe⁰ -Con-G also exhibited partialagonist activity. The partial agonists, as presently ascertained, aresummarized in Table III below. EC₅₀ and lC₅₀. values are reported inmicromolar concentrations. Standard errors of the mean are provided whenavailable.

                  TABLE III                                                       ______________________________________                                        Structure   EC.sub.50                                                                              % STI     IC.sub.50                                                                            % INH                                   ______________________________________                                        Phe.sup.0 -Con-G                                                                          6.8 ± 1.6                                                                           53 ± 17                                                                              6.1    51                                        tBu-Tyr.sup.0 -Con-G 1.0 69 1.5 29                                            Ser3-Con-G 8.4 ± 1.6 62 ± 4.90 3.6 ± 0.7 23 ± 3.6                 Ser(p) 3-Con-G 3,5 ± 1.9 68 ± 8.4 0.9 ± 0.09 34 ± 2.3                                                  Glu-Con-G 6.7 79 8.5 22                  Glu3,4,7,10,14- 0.9 33 5.4 70                                               ______________________________________                                    

EXAMPLE 4 Con G Derivatives Exhibiting Full Agonist Activity

The derivatives were tested for their effect on [³ H]MK-801 in theabsence of spermine to determine whether any of the compounds exhibitedagonistic properties for the NMDA receptor. All the compounds withagonistic activities are active in this assay.

As depicted in FIG. 10, Con-G(14-17) (inverted triangles), Tyr⁰-Glu-Con-G (filled triangles), D-Glu-Con-G (open triangles), andGlu-Con-G(12-17) (circles) stimulated [³ H]MK-801 binding in the absenceof spermine. These derivatives exhibited no inhibition ofspermine-induced [³ H]MK-801 binding.

EXAMPLE 5 Binding Site Studies.

As Con-G, ifenprodil also inhibits polyamine binding in anon-competitive manner. It does not appear, however, that Con-G acts atthe same site as ifenprodil since Con-G slightly increases, whileifenprodil decreases (Reynolds et al., supra ), [³ H]MK-801 binding inthe nominal absence of Gly and Glu. Further, ifenprodil inhibits Gluenhanced [³ H]MK-801 binding, while as shown in FIG. 1, and discussedbelow, even at very high concentrations, Con-G does not appear to affectGlu stimulation.

Spermine (at a concentration of up to 1000 μM) lacks the ability toreverse the antagonistic effects of both Con-G and Ala7-Con-G onNMDA-stimulated cyclic GMP formation. This observation is in agreementwith non-competitive inhibition seen in radioligand binding assays,indicating that the polyamines and Con-G do not compete at the samebinding site on the NMDA receptor complex.

Con-G does, however, inhibit Glu-Con-G stimulated [³ H]MK-801 binding.This suggests that derivatives of Con-G exhibiting agonist activity arebinding at the polyamine site. The results are depicted in FIG. 11. InFIG. 11, squares represent Glu-Con-G alone; triangles representGlu-Con-G with 1 μM Con-G; and circles represent Glu-Con-G with 2 μMCon-G.

Further evidence that the Con-G derivatives exhibiting agonist activityact at the polyamine site is given by their interactions with arcaine, acompetitive inhibitor of the polyamine site. The results withGlu-Con-G(12-17) are depicted in FIG. 12. In FIG. 12, squares representGlu-Con-G(12-17) stimulation of [³ H]MK-801 binding; triangles representthe effect of 5 μM arcaine on Glu-Con-G(12-17) stimulated [³ H]MK-801binding; and diamonds represent the effect of 10 μM arcaine onGlu-Con-G(12-17) stimulated [³ H]MK-801 binding.

FIG. 13 depicts the effect of increasing concentrations of Con-G uponGly- and Glu-stimulated [³ H]MK-801 binding to the brain membranepreparation (triangles represent Con-G plus 10 μM Glu; diamondsrepresent Con-G plus 10 μM Gly; and circles represent Con-G alone). Ascan be seen, Con-G has no affect upon Glu stimulation and, in fact, theGlu stimulation is additive with that produced by Con-G alone. Con-Gdoes slightly inhibit Gly stimulation at concentrations greater than 5μM. Radioligand binding studies with [³ H]CGP39653 confirmed that Con-Gand its derivative Ala7 had no actions at Glu binding site. To ascertainwhether Con-G acts as a competitive inhibitor at the Gly site, theeffect of 10 μM Con-G on Gly stimulated [³ H]MK-801 binding to themembrane preparation was determined. The results are depicted in FIG.14. In FIG. 14, triangles represent Gly alone; squares represent Glywith 10 μM Con-G. Con-G modestly inhibits Gly enhanced [³ H]MK-801binding by about 15% with no remarkable effect on its potency. Thedotted line represents a theoretical binding curve if the combination ofCon-G and Gly was additive. As can be seen in FIG. 14, Con-G does notinhibit the Gly stimulation in a manner consistent with competitiveinhibition. Therefore, Con-G does not appear to be a competitiveinhibitor of the Gly site.

Similar results were obtained with the derivative Ala7-Con-G. Ala7-Con-Gdid not affect Glu-mediated increases in [³ H]MK-801 binding, but didantagonize Gly-mediated increases in [³ H]MK-801 binding. The resultswith Ala7-Con-G were obtained in the presence (triangles) or absence(circles) of Gluate and .are presented in FIG. 15b. However, at highconcentrations, Con-G partially (˜65%) and Ala7-Con-G fully antagonizedthe binding of [³ H]MK-801 produced by Gly (10 μM) with IC₅₀ values of513±72 (n=3) and 858±133 nM (n=3), respectively. These results aredepicted in FIG. 15a. In FIG. 15a, circles represent Con-G; diamondsrepresent Ala7-Con-G; the dotted line represents maximum Gly stimulated[³ H]MK-801 binding; and dashed line represents baseline Gly stimulated[³ H]MK-801 binding. In this representative experiment, the IC₅₀ valuesof Ala7-Con-G and Con-G are 625 and 835 nM, respectively.

At a concentration of 30 nM, Ala7-Con-G reduced theconcentration-response curve of spermine to baseline values, whereasGly-stimulated [³ H]MK-801 binding was only reduced by about 20% (FIG.16: diamonds, Gly without Ala7-Con-G; circles, Gly with 30 nMAla7-Con-G). The EC₅₀. values of Gly in this experiment were 56 and 58nM in the presence and absence of Ala7, respectively. This experimentwas repeated with similar results. The IC₅₀ value of Ala7 (˜500 nM)required to reduce Gly stimulated [³ H]MK-801 binding was 10 times morethan that required to reduce spermine stimulated [³ H]MK-801 binding(˜45 nM).

Neither Con-G nor Ala7-Con-G affected the binding of [³ H]5,7-dichlorokynurenic acid (DCK), which is a specific radioligand for thestrychnine-insensitive Gly site on the NMDA receptor complex, [³ H]CGS39753, a radioligand specific for the Gluate site, or [³ H]Ifrenprodil, an NMDA antagonist acting at a polyamine-sensitive site.These results are summarized in Table IV below. IC₅₀ values are reportedin μM concentration.

                  TABLE IV                                                        ______________________________________                                        Example  [.sup.3 H]-CGS39753                                                                        [.sup.3 H]DCK                                                                           [.sup.3 H]IFENPRODIL                          ______________________________________                                        Con-G    μ5 ρM >5 μM  >5 μM                                        Ala7-Con-G >2.5 μM >2.5 μM 2.5 μM                                  ______________________________________                                    

In contrast, the inhibitory effects of Con-G (up to 2.5 μM) andAla7-ConG (up to 2.5 μM) on spermine-, Gly-, and spermine-Gly-enhancedincreases in [³ H]MK-801 binding were abolished in the presence of 10 μMGluate.

The inhibition of Gly effects by Ala7-Con-G and Con-G might be producedthrough the allosteric interaction of spermine associated site and Glysite on the NMDA receptor complex. Consistent with these conclusions,there is some evidence that has been reported suggesting that thepolyamines increase receptor response by increasing the apparentaffinity of Gly at the strychnine-insensitive Gly site. It is,therefore, possible that the modest reduction of Gly stimulation isattributable to Con-G inhibition of some remaining endogenous polyaminesin the washed brain membranes.

EXAMPLE 6 Con G Derivatives with Modified Helical Cores

The structure of the 17 amino acid Con-G isolated from snail wasdetermined by NMR, CD and IR to have a stiff helical core spanning aminoacid residues 7 to 13. This helical core is flanked by two flexible endpieces, a 6 amino acid long amino terminal piece and a 4 amino acid longcarboxy terminal piece. Extensive structure activity data on Con-Ganalogues which have a full spectrum of activities ranging fromantagonism through partial agonism/antagonism to full agonism wasobtained in order to determine the domains of the peptide responsiblefor either agonist or antagonist activity and to establish thedeterminants and amino acids required for those activities. The flexibleamino terminus is responsible for the NMDA antagonist or down regulatoryactivity of the peptide, whereas the flexible carboxy terminus isresponsible for the polyamine agonist activity. From this information,the desired level of agonist or antagonist activity may be designed intoshort peptide fragments in order to obtain agonists, partial agonists orantagonists with the desired level of stimulation or inhibition.

Exchanging the native helical core with different sequences was found tohave a profound impact on binding and activity measures. Con-G analogswere designed and synthesized with cores (linkers) containing compoundsknown to produce an alpha helix (SEQ ID NO:56, "alpha helix ConG"), a3₁₀ helix (SEQ ID NO:55, "3₁₀ helix ConG") or to disrupt the helicalstructures. The binding affinity as well as their agonist/antagonistactivity were measured. It was found that the two flexible end pieceshave to be oriented in a specific direction and point away from thehelix at a specific angle to have proper alignment and spacing forfunction.

ConG analogs with modified helical cores are shown in Table 1 as SEQ IDNOs. 55-64. In SEQ ID NO. 64, "B" represents amino isobutyvic acid. Theactivities of the analogs are illustrated in Table II. Additionally therelative helicity of each of the modified cores is represented on ascale of 1 to 5, from least helical to most helical, where 1=unordered,2=one turn, 3=loosened alpha or 3₁₀ helix, 4=stabilized α helix inwater, and 5=a full helix.

FIG. 20 is a graph illustrating the relative antagonism of ConG and theanalogs Ser¹⁴ -ConG, Ala⁷ -ConG and Ala⁷,10,14 -ConG. ConG and Ser¹⁴-ConG only antagonize the PA portion of the overall NMDA receptorstimulation; the others, Ala⁷ -ConG and Ala⁷,10 ,14 -ConG, antagonizethe PA portion of the Glu and Gly portions of the NMDA receptorstimulation. The results of the determination of the antagonism activitywith different analogs illustrates that the three dimensional structureof the core or linker is important for the type of antagonism exhibited.An alpha helical core will cause the analog to be a full NMDA receptorantagonist, capable of suppressing Glu, Gly, and PA stimulation, whereasa 3₁₀ helical core will cause the analog to be a selective pA antagonistsuppressing only the PA but not the Glu and Gly portions of the NMDAreceptor stimulation. The length and structure of the linker thus is onefeature of the peptide which is important for activity. As few as one totwo turns, or as many as four to five turns, in the linker helix aresufficient for activity. The 3₁₀ helix includes about three amino acidsper turn. The alpha helix includes approximately 3.7 amino acids perturn.

In summary, the data makes it is clear that Con-G is acting as anantagonist to polyamine stimulated binding at a novel and previouslyunrecognized allosteric modulatory/regulatory site on the NMDA receptor.

EXAMPLE 7 Inhibition of cGMP Formation

Cyclic GMP assays

Experiments were carried out after 8-day plating. The culture disheswere washed twice with 0.5 ml of Lock's buffer without magnesium (154 mM

NaCl, 5.6 mM KCl, 2.3 mM CaCl, 5.6 mM glucose, 8.6 mM HEPES, pH 7.4).Cells were preincubated in the same medium for 15 min at 25° C. in thepresence and absence of antagonists or/and spermine. Cells were thenincubated for 3 min in the presence and absence of NMDA (100 μM) orkainate (50 μM). The incubation was terminated by rapid removal ofmedium followed by addition of 0.2 ml of 0.4 M HClO₄. Cells were scrapedoff the dishes and sonicated. Aliquots of the homogenates wereneutralized with KOH, centrifuged to remove KClO₄, and adjusted to pH 6with acetic acid. Cyclic GMP content was determined using a commerciallyavailable radioimmunoassay kit (Biomedical Technologies, Inc.,Stoughton, Mass., U.S.A.) . The results are presented as the means withS.E.M. of assays in triplicate, and the data are expressed as picomolesof cyclic GMP per mg of protein.

Results

Ala7-Con-G inhibited NMDA-stimulated increases in cyclic GMP formationin primary cultures of granule cells with an IC₅₀ value of 77±7.5 nM(n=4), 4-fold (p<0.01) greater than that of Con-G (IC₅₀ 300±30 nM, n=5)under the same conditions, without altering kainate (50 μM)- mediatedeffects (up to 2. 5 μM) . The results are depicted in FIG. 17. In thisrepresentative experiment, Con-G (squares) and Ala7-Con-G (circles)inhibited NMDA (100 μM) stimulated increases in cyclic GMP with IC₅₀values of 332 and 78 nM, respectively (data represent the means ± SEM oftriplicate values).

EXAMPLE 8 Protection Against Neurotoxicity

Evaluation of Neurotoxicity

Preparation of Cerebellar Granule Cells

Primary cultures of cerebellar granule cells were prepared from 6-8 dayold Sprague-Dawley rats by the method of Gallo et al., "Selectiverelease of glutamate from cerebellar granule cells differentiating inculture," Proc. Nat'l Acad. Sci. USA, 29:7010-7023, 1982, incorporatedherein by reference. Dissociated cells were resuspended in Eagle's BasalMedium (Gibco, Grand Island, N.Y.) with 10% fetal calf serum (QualityBiological, Inc., Gaithersburg, MD), 2 mM 1-glutamine, 0.1 mg/mlgentamicin, and 25 mM KCl. (All remaining cell culture reagents wereobtained from Sigma, Co., St. Louis, Mo.) The cells were seeded in 22-35mm poly-L-lysine (10 μml) coated dishes at a density of 1.8-3.8×10⁵cells/cm². Cytosine arabinoside (10 μM) was added 18 to 24 hours afterplating to inhibit the growth of non-neuronal cells. Cultures generatedby this method contained ≧90% granule cell neurons. The medium was notchanged during the culture period. Cells were incubated at 37° C. inhumidified 95% air/5% CO₂ atmosphere.

Determination of Neurotoxicity

Experiments were performed using cells cultured for 9 days. The culturemedium was removed and the granule cells were washed twice with 1.5 mlof incubation buffer (160.6 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl ₂, and 8.6mM HEPES, Ph 7.4-Locke buffer without magnesium or glucose). The cellswere preincubated in 1 ml of buffer for 25 minutes at 37° C. Following achange of buffer, test compounds were added to the appropriate culturesand incubated for an additional 15 minutes (37° C.). The buffer waschanged again, and Glu (100 μM) was added together with Con-G (0.1-5μM), MK-801 (10 μM), or vehicle. Thirty minutes later, the buffer wasremoved, the cells washed twice with 1.5 ml of complete Locke buffer(154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl₂, 5.6 mM glucose, 8.6 mM HEPES,1-0 Mm MgCl₂, pH 7.4), and the cells incubated for 18-24 hours in 1 mlof culture medium. Cell death was determined using trypan blue. Celldeath and neuroprotection were calculated by the following equations:

    % cytotoxicity=D/(D+L)×100;

    % protection=100-(% cytotoxicity)

where "D" equals the number of dead cells counted in three differentrandom fields using 400× bright field microscopy, and "L" equals thenumber of live cells counted in the same three fields.

FIG. 18 documents that Con-G may be used to protect cells fromexcitotoxic death (resulting from excess Ca⁺⁺ influx) induced by Gluover-stimulation of the NMDA receptor. In FIG. 18, the protective effectof Con-G and MK-801 on cerebellar granule cell cultures was measured inresponse to a neurotoxicity induced with a 100 μM concentration of Glu.Con-G's action at the Con-G site is sufficient to reduce the flow ofCa⁺⁺ to substantially protect the cells from excitotoxic death. In fact,the protection achieved with 5 μM Con-G is comparable to that obtainedwith 10 μM MK-801. Since Con-G does not actually block the channel, itis not expected to produce the dramatic long term effects seen with thechannel blocking compounds. Thus, Con-G's action in allostericallymodulating the response of the polyamine site acts to modulate theoverall response of the NMDA receptor.

EXAMPLE 9 Determination of Subtype Specific Binding.

The various compounds can be utilized in assays for determiningcompounds which bind the novel Con-G site, or portions thereof. Forexample, some of the derivatives prepared bind at the Con site alone,whereas others bind to the polyamine site alone. Still others, likeCon-G, bind to both. Labelled compounds representative of eachgroup--full antagonists, partial agonists, and full agonists--can beused to ascertain other compounds with similar binding characteristics.

Inhibition of Polyamine Stimulation of the NR1/NR2b Subtype

Con-G was found to be specific for one subtype of the NMDA receptor, theNR1/NR2b subtype. As described in Kutsuwada et al., Nature, 358:36-41(1992), the disclosure of which is incorporated herein by reference,NR2a, NR2b and NR2c subunit-specific mRNAs were synthesized in vitrofrom cloned cDNA and injected in Xenopus oocytes together with the NR1subunit-specific MnRNA. The peak inward currents obtained in normal frogRinger's solution at -70mV membrane potential were 300 nA in response to10 μM L-Glu plus 10 μM Gly and 100 μM NMDA plus 10 μM Gly. They wereabout the same for oocytes injected with all three subunit combinations.The current amplitudes were much larger than those for oocytes implantedwith the NR1 subunit alone. Also oocytes injected with the NR2subunit-specific mRNAs alone exhibited no detectable response (<1 nA).

Neither 100 μM kainate nor 100 μM AMPA evoked a measurable response ofoocytes injected with the NR1 and NR2 subunits. Addition of polyamineshad no effect on inward current in the NR1/NR2a and NR1/NR2c subunitinjected oocytes, but increased the amplitude in the NR1/NR2btransformed oocytes by about 30%. This data is consistent with thefinding that polyamines act on the NR1/NR2b subtype of the NMDAreceptor. Igarashi et al., J. Pharm. Exp. Ther., 272:1101-1109 (1995).

Current responses of polyamine stimulated Xenopus oocytes injected withthe heteromeric NRl1/NR2a, NR1/NR2b, NR1/NR2c NMDA receptor subunits toConG were examined. As illustrated in FIG. 19, subunit-specific effectsof spermine and ConG were measured by inducing currents with L-Glu andGly (10 μM each) in oocytes voltage-clamped at -70 μM and expressing theNR1/NR2a, NR1/NR2b and the NR1/NR2c receptors in the presence of 10 μMspermine. 3 μM ConG was applied during the times shown by the horizontalbar in FIG. 19. Addition of ConG at 3 μM had no effect on currentsthrough the NR1/NR2a and NR1/NR2c receptor, but inhibited the polyaminestimulation on the NR1/NR2b receptor subtype. This shows that ConG isthe first and only polyamine specific inhibitor in as far as itscapability to inhibit polyamine stimulated ion flow.

ConG and analogs and mimetics thereof thus can be used as probes forexamining the physiology and role of polyamines on the NR1/NR2b subtypeof the NMDA receptor in neuropsychoparmacological disorders, and for thedevelopment of clinically effective drugs. Modified peptides and mimeticmolecules can be designed which have a preselected affinity and activityas well as which have enhanced stability and bioavailability.

Although the invention has been described with reference to Con-G andspecific Con-G derivatives, the details should not be construed aslimitations on the invention. Rather, various equivalents,modifications, and other derivatives as would be obvious to one skilledin the art upon learning of the invention and without departing from thespirit and scope of the invention as presented in the appended claimsare included. All references cited herein are hereby incorporated hereinby reference.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 65                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                            amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 5 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 8 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 11 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 15 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 18 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Tyr Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xa - #a Leu Ile Arg Xaa          1               5   - #                10  - #                15               - - Lys Ser Asn                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 1 is                                  acetylated"                                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Gly Glu Glu Glu Leu Gln Glu Asn Gln Glu Le - #u Ile Arg Glu Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 18 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Tyr Gly Glu Glu Glu Leu Gln Glu Asn Gln Gl - #u Leu Ile Arg Glu          1               5   - #                10  - #                15               - - Lys Ser Asn                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 6 has an                              amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Ile Arg Glu Lys Ser Asn                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 9 has an                              amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Gln Glu Leu Ile Arg Glu Lys Ser Asn                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 12 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Gln Glu Asn Gln Glu Leu Ile Arg Glu Lys Se - #r Asn                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 16                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 16 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Glu Glu Glu Leu Gln Glu Asn Gln Glu Leu Il - #e Arg Glu Lys Ser          1               5   - #                10  - #                15               - - Asn                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 2 is                                  D-glutamate - #(D-Glu)"                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  D-glutamate - #(D-Glu)"                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  D-glutamate - #(D-Glu)"                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  D-glutamate - #(D-Glu)"                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 D-glutamate - #(D-Glu)"                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 D-glutamate - #(D-Glu)"                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Gly Xaa Xaa Xaa Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 21                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 21 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Gly Glu Xaa Xaa Tyr Gln Lys Met Leu Xaa As - #n Leu Arg Xaa Ala          1               5   - #                10  - #                15               - - Glu Val Lys Lys Asn Ala                                                                  20                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 1 is                                  acetylated"                                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 5 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 8 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 11 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 15 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 18 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Tyr Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xa - #a Leu Ile Arg Xaa          1               5   - #                10  - #                15               - - Lys Ser Asn                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 5 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 8 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 11 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 15 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 18 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Asn Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xa - #a Leu Ile Arg Xaa          1               5   - #                10  - #                15               - - Lys Ser Asn                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 1 has                                 N-acetylgluc - #osamine"                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 5 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 8 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 11 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 18 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Asn Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xa - #a Leu Ile Arg Xaa          1               5   - #                10  - #                15               - - Lys Ser Asn                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 5 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 8 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 11 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 18 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Phe Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xa - #a Leu Ile Arg Xaa          1               5   - #                10  - #                15               - - Lys Ser Asn                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 1 is                                  phosphochlor - #inated"                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 5 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 8 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 11 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 18 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Phe Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xa - #a Leu Ile Arg Xaa          1               5   - #                10  - #                15               - - Lys Ser Asn                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 1 is                                  phosphorylat - #ed"                                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 5 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 8 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 11 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 18 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Tyr Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xa - #a Leu Ile Arg Xaa          1               5   - #                10  - #                15               - - Lys Ser Asn                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 1 is                                  methylated"                                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 5 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 8 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 11 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 18 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Tyr Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xa - #a Leu Ile Arg Xaa          1               5   - #                10  - #                15               - - Lys Ser Asn                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 1 is                                  t-butylated"                                                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 5 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 8 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 11 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 18 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Tyr Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xa - #a Leu Ile Arg Xaa          1               5   - #                10  - #                15               - - Lys Ser Asn                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 5 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 8 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 11 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 18 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Ser Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xa - #a Leu Ile Arg Xaa          1               5   - #                10  - #                15               - - Lys Ser Asn                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - Gly Glu Ala Xaa Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Gly Glu Ser Xaa Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  phosphorylat - #ed"                                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Gly Glu Ser Xaa Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - Gly Glu Tyr Xaa Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - Gly Glu Xaa Glu Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - Gly Glu Xaa Ala Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - Gly Glu Xaa Ser Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  phosphorylat - #ed"                                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamte (Gla)"                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - Gly Glu Xaa Ser Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - Gly Glu Xaa Xaa Tyr Gln Xaa Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - Gly Glu Xaa Xaa Leu Gln Tyr Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - Gly Glu Xaa Xaa Leu Gln Ala Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - Gly Glu Xaa Xaa Leu Gln Ser Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  phosphorylat - #ed"                                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - Gly Glu Xaa Xaa Leu Gln Ser Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Ala Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Ser Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 phosphorylat - #ed"                                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Ser Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - - Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Ala Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - - Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Ser Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 phosphorylat - #ed"                                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - - Gly Glu Xaa Xaa Leu Gln Xaa Asn Gln Xaa Le - #u Ile Arg Ser Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - - Gly Glu Xaa Xaa Leu Gln Glu Asn Gln Glu Le - #u Ile Arg Glu Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - - Gly Glu Xaa Xaa Leu Gln Ala Asn Gln Ala Le - #u Ile Arg Ala Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 6 has an                              amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - - Ile Arg Glu Ala Ser Asn                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 6 has an                              amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - - Ile Ala Glu Lys Ser Asn                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 6 has an                              amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                              - - Ile Ala Glu Ala Ser Asn                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 6 has an                              amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                              - - Ile Arg Xaa Lys Ser Asn                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 5 has an                              amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                              - - Arg Ser Xaa Asn Lys                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 5 has an                              amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                              - - Arg Xaa Lys Ser Asn                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 1 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 has an                              amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                              - - Xaa Lys Ser Asn                                                          1                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 has an                              amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                              - - Ile Arg Xaa Lys                                                          1                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 has an                              amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                              - - Lys Ser Asn                                                              1                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 5 has an                              amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                              - - Gly Glu Xaa Xaa Leu                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  iodinated - #(cold)"                                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                              - - Gly Glu Xaa Xaa Leu Gln Tyr Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  di-iodinated - # (cold)"                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                              - - Gly Glu Xaa Xaa Leu Gln Tyr Asn Gln Xaa Le - #u Ile Arg Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                              - - Gly Glu Xaa Xaa Leu Gln Ser Asn Val Ser Gl - #n Ile Arg Ala Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                              - - Gly Glu Xaa Xaa Leu Gln Ala Ala Leu Ala Le - #u Ile Arg Ala Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 6 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 9 has an                              amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                              - - Gly Glu Xaa Xaa Leu Xaa Lys Ser Asn                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 8 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 11 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                              - - Gly Glu Xaa Xaa Leu Gly Gly Xaa Lys Ser As - #n                          1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 10 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 13 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                              - - Gly Glu Xaa Xaa Leu Gly Gly Gly Gly Xaa Ly - #s Ser Asn                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 8 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 11 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                              - - Gly Glu Xaa Xaa Leu Ala Ala Xaa Lys Ser As - #n                          1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 9 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 12 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                              - - Gly Glu Xaa Xaa Leu Ala Ala Ala Xaa Lys Se - #r Asn                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 12 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 15 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                              - - Gly Glu Xaa Xaa Leu Gln Ala Ala Ala Ala Al - #a Xaa Lys Ser Asn          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 14 is                                 gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 17 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                              - - Gly Glu Xaa Xaa Leu Gln Ala Ala Ala Ala Al - #a Ala Ala Xaa Lys          1               5   - #                10  - #                15               - - Ser Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 6 is                                  amino isi - #butryic acid"                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 7 is                                  amino isi - #butryic acid"                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 8 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/note= "Amino acid 11 has an                             amidated - #terminus"                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                              - - Gly Glu Xaa Xaa Leu Xaa Xaa Xaa Lys Ser As - #n                          1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 3 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 4 is                                  gamma-carbox - #yglutamate (Gla)"                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "Amino acid 5 is cyclic                           and has - #an amidated terminus"                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                              - - Gly Glu Xaa Xaa Leu                                                      1               5                                                            __________________________________________________________________________

What is claimed is:
 1. A method for treating excitotoxicity resultingfrom over-stimulation of the NMDA receptor which comprises administeringto a patient in need thereof a therapeutically effective amount ofConantokin-G or a pharmaceutically acceptable salt thereof.
 2. Themethod of claim 1, wherein said excitotoxicity is associated withepilepsy or epileptic seizure.